Novel crispr enzymes and systems

ABSTRACT

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Methods for making and using and uses of such systems, methods, and compositions and products from such methods and uses are also disclosed and claimed.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. application Ser. No.16/400,026, filed Apr. 30, 2019, which is a continuation of U.S.application Ser. No. 15/844,608, filed Dec. 17, 2017, now U.S. Pat. No.10,648,020 that issued on May 12, 2020, which is a continuation-in-partapplication of international patent application Serial No.PCT/US2016/038181, filed Jun. 17, 2016, which published as PCTPublication No. WO2016/205711 on Dec. 22, 2016, and which claims benefitof and priority to U.S. Provisional Application No. 62/181,739, filedJun. 18, 2015, U.S. Provisional Application No. 62/193,507, filed Jul.16, 2015, U.S. Provisional Application No. 62/201,542, filed Aug. 5,2015, U.S. Provisional Application No. 62/205,733, filed Aug. 16, 2015,U.S. Provisional Application No. 62/232,067, filed Sep. 24, 2015, andEuropean Application No. 16150428.7, filed Jan. 7, 2016, and is acontinuation-in-part of U.S. application Ser. No. 14/975,085, filed Dec.18, 2015, now U.S. Pat. No. 9,790,490, that issued on Oct. 17, 2017.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.MH100706, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 20, 2022, isnamed 114203-6003_Sequence_Listing.TXT and is 2,446,134 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as perturbation of gene transcripts or nucleic acidediting, that may use vector systems related to Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that employ novelstrategies and molecular mechanisms and are affordable, easy to set up,scalable, and amenable to targeting multiple positions within theeukaryotic genome. This would provide a major resource for newapplications in genome engineering and biotechnology.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.The CRISPR-Cas system loci has more than 50 gene families and there isno strictly universal genes indicating fast evolution and extremediversity of loci architecture. So far, adopting a multi-prongedapproach, there is comprehensive cas gene identification of about 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in which these systems are broadlydivided into two classes, Class 1 with multisubunit effector complexesand Class 2 with single-subunit effector modules exemplified by the Cas9protein. Novel effector proteins associated with Class 2 CRISPR-Cassystems may be developed as powerful genome engineering tools and theprediction of putative novel effector proteins and their engineering andoptimization is important.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There exists a pressing need for alternative and robust systems andtechniques for targeting nucleic acids or polynucleotides (e.g. DNA orRNA or any hybrid or derivative thereof) with a wide array ofapplications. This invention addresses this need and provides relatedadvantages. Adding the novel DNA or RNA-targeting systems of the presentapplication to the repertoire of genomic and epigenomic targetingtechnologies may transform the study and perturbation or editing ofspecific target sites through direct detection, analysis andmanipulation. To utilize the DNA or RNA-targeting systems of the presentapplication effectively for genomic or epigenomic targeting withoutdeleterious effects, it is critical to understand aspects of engineeringand optimization of these DNA or RNA targeting tools.

The invention provides a method of modifying sequences associated withor at a target locus of interest, the method comprising delivering tosaid locus a non-naturally occurring or engineered compositioncomprising a putative Type V CRISPR-Cas loci effector protein and one ormore nucleic acid components, wherein the effector protein forms acomplex with the one or more nucleic acid components and upon binding ofthe said complex to the locus of interest the effector protein inducesthe modification of the sequences associated with or at the target locusof interest. In a preferred embodiment, the modification is theintroduction of a strand break. In a preferred embodiment, the sequencesassociated with or at the target locus of interest comprises DNA and theeffector protein is encoded by a subtype V-A CRISPR-Cas loci or asubtype V-B CRISPR-Cas loci.

It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPRprotein Cas protein and CRISPR Cas are generally used interchangeablyand at all points of reference herein refer by analogy to novel CRISPReffector proteins further described in this application, unlessotherwise apparent, such as by specific reference to Cas9. The CRISPReffector proteins described herein are preferably Cpf1 effectorproteins.

The invention provides a method of modifying sequences associated withor at a target locus of interest, the method comprising delivering tosaid sequences associated with or at the locus a non-naturally occurringor engineered composition comprising a Cpf1 loci effector protein andone or more nucleic acid components, wherein the Cpf1 effector proteinforms a complex with the one or more nucleic acid components and uponbinding of the said complex to the locus of interest the effectorprotein induces the modification of the sequences associated with or atthe target locus of interest. In a preferred embodiment, themodification is the introduction of a strand break. In a preferredembodiment the Cpf1 effector protein forms a complex with one nucleicacid component; advantageously an engineered or non-naturally occurringnucleic acid component. The induction of modification of sequencesassociated with or at the target locus of interest can be Cpf1 effectorprotein-nucleic acid guided. In a preferred embodiment the one nucleicacid component is a CRISPR RNA (crRNA). In a preferred embodiment theone nucleic acid component is a mature crRNA or guide RNA, wherein themature crRNA or guide RNA comprises a spacer sequence (or guidesequence) and a direct repeat sequence or derivatives thereof. In apreferred embodiment the spacer sequence or the derivative thereofcomprises a seed sequence, wherein the seed sequence is critical forrecognition and/or hybridization to the sequence at the target locus. Ina preferred embodiment, the seed sequence of a FnCpf1 guide RNA isapproximately within the first 5 nt on the 5′ end of the spacer sequence(or guide sequence). In a preferred embodiment the strand break is astaggered cut with a 5′ overhang. In a preferred embodiment, thesequences associated with or at the target locus of interest compriselinear or super coiled DNA.

Aspects of the invention relate to Cpf1 effector protein complexeshaving one or more non-naturally occurring or engineered or modified oroptimized nucleic acid components. In a preferred embodiment the nucleicacid component of the complex may comprise a guide sequence linked to adirect repeat sequence, wherein the direct repeat sequence comprises oneor more stem loops or optimized secondary structures. In a preferredembodiment, the direct repeat has a minimum length of 16 nts and asingle stem loop. In further embodiments the direct repeat has a lengthlonger than 16 nts, preferrably more than 17 nts, and has more than onestem loop or optimized secondary structures. In a preferred embodimentthe direct repeat may be modified to comprise one or moreprotein-binding RNA aptamers. In a preferred embodiment, one or moreaptamers may be included such as part of optimized secondary structure.Such aptamers may be capable of binding a bacteriophage coat protein.The bacteriophage coat protein may be selected from the group comprisingQβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1,TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r,7s and PRR1. In a preferred embodiment the bacteriophage coat protein isMS2. The invention also provides for the nucleic acid component of thecomplex being 30 or more, 40 or more or 50 or more nucleotides inlength.

The invention provides methods of genome editing wherein the methodcomprises two or more rounds of Cpf1 effector protein targeting andcleavage. In certain embodiments, a first round comprises the Cpf1effector protein cleaving sequences associated with a target locus faraway from the seed sequence and a second round comprises the Cpf1effector protein cleaving sequences at the target locus. In preferredembodiments of the invention, a first round of targeting by a Cpf1effector protein results in an indel and a second round of targeting bythe Cpf1 effector protein may be repaired via homology directed repair(HDR). In a most preferred embodiment of the invention, one or morerounds of targeting by a Cpf1 effector protein results in staggeredcleavage that may be repaired with insertion of a repair template.

The invention provides methods of genome editing or modifying sequencesassociated with or at a target locus of interest wherein the methodcomprises introducing a Cpf1 effector protein complex into any desiredcell type, prokaryotic or eukaryotic cell, whereby the Cpf1 effectorprotein complex effectively functions to integrate a DNA insert into thegenome of the eukaryotic or prokaryotic cell. In preferred embodiments,the cell is a eukaryotic cell and the genome is a mammalian genome. Inpreferred embodiments the integration of the DNA insert is facilitatedby non-homologous end joining (NHEJ)-based gene insertion mechanisms. Inpreferred embodiments, the DNA insert is an exogenously introduced DNAtemplate or repair template. In one preferred embodiment, theexogenously introduced DNA template or repair template is delivered withthe Cpf1 effector protein complex or one component or a polynucleotidevector for expression of a component of the complex. In a more preferredembodiment the eukaryotic cell is a non-dividing cell (e.g. anon-dividing cell in which genome editing via HDR is especiallychallenging). In preferred methods of genome editing in human cells, theCpf1 effector proteins may include but are not limited to FnCpf1, AsCpf1and LbCpf1 effector proteins.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a C2c1 loci effectorprotein and one or more nucleic acid components, wherein the C2c1effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

In such methods the target locus of interest may be comprised in a DNAmolecule in vitro. In a preferred embodiment the DNA molecule is aplasmid.

In such methods the target locus of interest may be comprised in a DNAmolecule within a cell. The cell may be a prokaryotic cell or aeukaryotic cell. The cell may be a mammalian cell. The mammalian cellmany be a non-human primate, bovine, porcine, rodent or mouse cell. Thecell may be a non-mammalian eukaryotic cell such as poultry, fish orshrimp. The cell may also be a plant cell. The plant cell may be of acrop plant such as cassava, corn, sorghum, wheat, or rice. The plantcell may also be of an algae, tree or vegetable. The modificationintroduced to the cell by the present invention may be such that thecell and progeny of the cell are altered for improved production ofbiologic products such as an antibody, starch, alcohol or other desiredcellular output. The modification introduced to the cell by the presentinvention may be such that the cell and progeny of the cell include analteration that changes the biologic product produced.

The invention provides a method of modifying a target locus of interest,the method comprising delivering to said locus a non-naturally occurringor engineered composition comprising a Type VI CRISPR-Cas loci effectorprotein and one or more nucleic acid components, wherein the effectorprotein forms a complex with the one or more nucleic acid components andupon binding of the said complex to the locus of interest the effectorprotein induces the modification of the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak.

In a preferred embodiment, the target locus of interest comprises DNA.

In such methods the target locus of interest may be comprised in a DNAmolecule within a cell. The cell may be a prokaryotic cell or aeukaryotic cell. The cell may be a mammalian cell. The mammalian cellmany be a non-human mammal, e.g., primate, bovine, ovine, porcine,canine, rodent, Leporidae such as monkey, cow, sheep, pig, dog, rabbit,rat or mouse cell. The cell may be a non-mammalian eukaryotic cell suchas poultry bird (e.g., chicken), vertebrate fish (e.g., salmon) orshellfish (e.g., oyster, claim, lobster, shrimp) cell. The cell may alsobe a plant cell. The plant cell may be of a monocot or dicot or of acrop or grain plant such as cassava, corn, sorghum, soybean, wheat, oator rice. The plant cell may also be of an algae, tree or productionplant, fruit or vegetable (e.g., trees such as citrus trees, e.g.,orange, grapefruit or lemon trees; peach or nectarine trees; apple orpear trees; nut trees such as almond or walnut or pistachio trees;nightshade plants; plants of the genus Brassica; plants of the genusLactuca; plants of the genus Spinacia; plants of the genus Capsicum;cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower,tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry,raspberry, blackberry, grape, coffee, cocoa, etc).

In any of the described methods the target locus of interest may be agenomic or epigenomic locus of interest. In any of the described methodsthe complex may be delivered with multiple guides for multiplexed use.In any of the described methods more than one protein(s) may be used.

In preferred embodiments of the invention, biochemical or in vitro or invivo cleavage of sequences associated with or at a target locus ofinterest results without a putative transactivating crRNA (tracr RNA)sequence, e.g. cleavage by an FnCpf1 effector protein. In otherembodiments of the invention, cleavage may result with a putativetransactivating crRNA (tracr RNA) sequence, e.g. cleavage by otherCRISPR family effector proteins, however after evaluation of the FnCpf1locus, Applicants concluded that target DNA cleavage by a Cpf1 effectorprotein complex does not require a tracrRNA. Applicants determined thatCpf1 effector protein complexes comprising only a Cpf1 effector proteinand a crRNA (guide RNA comprising a direct repeat sequence and a guidesequence) were sufficient to cleave target DNA. Accordingly, theinvention provides methods of modifying a target locus of interest asdescribed herein above, wherein the effector protein is a Cpf1 proteinand the effector protein complexes with the target sequence without thepresence of a tracr.

In any of the described methods the effector protein (e.g., Cpf1) andnucleic acid components may be provided via one or more polynucleotidemolecules encoding the protein and/or nucleic acid component(s), andwherein the one or more polynucleotide molecules are operably configuredto express the protein and/or the nucleic acid component(s). The one ormore polynucleotide molecules may comprise one or more regulatoryelements operably configured to express the protein and/or the nucleicacid component(s). The one or more polynucleotide molecules may becomprised within one or more vectors. The invention comprehends suchpolynucleotide molecule(s), for instance such polynucleotide moleculesoperably configured to express the protein and/or the nucleic acidcomponent(s), as well as such vector(s).

In any of the described methods the strand break may be a single strandbreak or a double strand break.

Regulatory elements may comprise inducible promotors. Polynucleotidesand/or vector systems may comprise inducible systems.

In any of the described methods the one or more polynucleotide moleculesmay be comprised in a delivery system, or the one or more vectors may becomprised in a delivery system.

In any of the described methods the non-naturally occurring orengineered composition may be delivered via liposomes, particles (e.g.nanoparticles), exosomes, microvesicles, a gene-gun or one or morevectors, e.g., nucleic acid molecule or viral vectors.

The invention also provides a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.

The invention also provides a vector system comprising one or morevectors, the one or more vectors comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.

The invention also provides a delivery system comprising one or morevectors or one or more polynucleotide molecules, the one or more vectorsor polynucleotide molecules comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.

The invention also provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in atherapeutic method of treatment. The therapeutic method of treatment maycomprise gene or genome editing, or gene therapy.

The invention also encompasses computational methods and algorithms topredict new Class 2 CRISPR-Cas systems and identify the componentstherein.

The invention also provides for methods and compositions wherein one ormore amino acid residues of the effector protein may be modified, e,g,an engineered or non-naturally-occurring effector protein or Cpf1. In anembodiment, the modification may comprise mutation of one or more aminoacid residues of the effector protein. The one or more mutations may bein one or more catalytically active domains of the effector protein. Theeffector protein may have reduced or abolished nuclease activitycompared with an effector protein lacking said one or more mutations.The effector protein may not direct cleavage of one or other DNA or RNAstrand at the target locus of interest. The effector protein may notdirect cleavage of either DNA or RNA strand at the target locus ofinterest. In a preferred embodiment, the one or more mutations maycomprise two mutations. In a preferred embodiment the one or more aminoacid residues are modified in a Cpf1 effector protein, e,g, anengineered or non-naturally-occurring effector protein or Cpf1. In apreferred embodiment the Cpf1 effector protein is a FnCpf1 effectorprotein. In a preferred embodiment, the one or more modified or mutatedamino acid residues are D917A, E1006A or D1255A with reference to theamino acid position numbering of the FnCpf1 effector protein. In furtherpreferred embodiments, the one or more mutated amino acid residues areD908A, E993A, D1263A with reference to the amino acid positions inAsCpf1 or LbD832A, E925A, D947A or D1180A with reference to the aminoacid positions in LbCpf1.

The invention also provides for the one or more mutations or the two ormore mutations to be in a catalytically active domain of the effectorprotein comprising a RuvC domain. In some embodiments of the inventionthe RuvC domain may comprise a RuvCI, RuvCII or RuvCIII domain, or acatalytically active domain which is homologous to a RuvCI, RuvCII orRuvCIII domain etc or to any relevant domain as described in any of theherein described methods. The effector protein may comprise one or moreheterologous functional domains. The one or more heterologous functionaldomains may comprise one or more nuclear localization signal (NLS)domains. The one or more heterologous functional domains may comprise atleast two or more NLS domains. The one or more NLS domain(s) may bepositioned at or near or in promixity to a terminus of the effectorprotein (e.g., Cpf1) and if two or more NLSs, each of the two may bepositioned at or near or in promixity to a terminus of the effectorprotein (e.g., Cpf1) The one or more heterologous functional domains maycomprise one or more transcriptional activation domains. In a preferredembodiment the transcriptional activation domain may comprise VP64. Theone or more heterologous functional domains may comprise one or moretranscriptional repression domains. In a preferred embodiment thetranscriptional repression domain comprises a KRAB domain or a SIDdomain (e.g. SID4X). The one or more heterologous functional domains maycomprise one or more nuclease domains. In a preferred embodiment anuclease domain comprises Fok1.

The invention also provides for the one or more heterologous functionaldomains to have one or more of the following activities: methylaseactivity, demethylase activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, nuclease activity,single-strand RNA cleavage activity, double-strand RNA cleavageactivity, single-strand DNA cleavage activity, double-strand DNAcleavage activity and nucleic acid binding activity. At least one ormore heterologous functional domains may be at or near theamino-terminus of the effector protein and/or wherein at least one ormore heterologous functional domains is at or near the carboxy-terminusof the effector protein. The one or more heterologous functional domainsmay be fused to the effector protein. The one or more heterologousfunctional domains may be tethered to the effector protein. The one ormore heterologous functional domains may be linked to the effectorprotein by a linker moiety.

The invention also provides for the effector protein (e.g., a Cpf1)comprising an effector protein (e.g., a Cpf1) from an organism from agenus comprising Streptococcus, Campylobacter, Nitratifractor,Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter,Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter,Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium,Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella,Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas,Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio,Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus,Methylobacterium or Acidaminococcus.

The invention also provides for the effector protein (e.g., a Cpf1)comprising an effector protein (e.g., a Cpf1) from an organism from S.mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C.jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S.carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L.ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii.

The effector protein may comprise a chimeric effector protein comprisinga first fragment from a first effector protein (e.g., a Cpf1) orthologand a second fragment from a second effector (e.g., a Cpf1) proteinortholog, and wherein the first and second effector protein orthologsare different. At least one of the first and second effector protein(e.g., a Cpf1) orthologs may comprise an effector protein (e.g., a Cpf1)from an organism comprising Streptococcus, Campylobacter,Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria,Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus,Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria,Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium,Leptotrichia, Francisella, Legionella, Alicyclobacillus,Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes,Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium orAcidaminococcus; e.g., a chimeric effector protein comprising a firstfragment and a second fragment wherein each of the first and secondfragments is selected from a Cpf1 of an organism comprisingStreptococcus, Campylobacter, Nitratifractor, Staphylococcus,Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum,Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium,Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae,Clostridiaridium, Leptotrichia, Francisella, Legionella,Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella,Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum,Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium orAcidaminococcus wherein the first and second fragments are not from thesame bacteria; for instance a chimeric effector protein comprising afirst fragment and a second fragment wherein each of the first andsecond fragments is selected from a Cpf1 of S. mutans, S. agalactiae, S.equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N.salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides,N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C.difficile, C. tetani, C. sordellii; Francisella tularensis 1, Prevotellaalbensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens and Porphyromonas macacae, whereinthe first and second fragments are not from the same bacteria.

In preferred embodiments of the invention the effector protein isderived from a Cpf1 locus (herein such effector proteins are alsoreferred to as “Cpf1p”), e.g., a Cpf1 protein (and such effector proteinor Cpf1 protein or protein derived from a Cpf1 locus is also called“CRISPR enzyme”). Cpf1 loci include but are not limited to the Cpf1 lociof bacterial species listed in FIG. 64 . In a more preferred embodiment,the Cpf1p is derived from a bacterial species selected from Francisellatularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1,Butyrivibrio proteoclasticus, Peregrinibacteria bacteriumGW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithellasp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020,Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxellabovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006,Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonasmacacae. In certain embodiments, the Cpf1p is derived from a bacterialspecies selected from Acidaminococcus sp. BV3L6, Lachnospiraceaebacterium MA2020. In certain embodiments, the effector protein isderived from a subspecies of Francisella tularensis 1, including but notlimited to Francisella tularensis subsp. Novicida.

In further embodiments of the invention a protospacer adjacent motif(PAM) or PAM-like motif directs binding of the effector protein complexto the target locus of interest. In a preferred embodiment of theinvention, the PAM is 5′ TTN, where N is A/C/G or T and the effectorprotein is FnCpf1p. In another preferred embodiment of the invention,the PAM is 5′ TTTV, where V is A/C or G and the effector protein isAsCpf1, LbCpf1 or PaCpf1p. In certain embodiments, the PAM is 5′ TTN,where N is A/C/G or T, the effector protein is FnCpf1p, and the PAM islocated upstream of the 5′ end of the protospacer. In certainembodiments of the invention, the PAM is 5′ CTA, where the effectorprotein is FnCpf1p, and the PAM is located upstream of the 5′ end of theprotospacer or the target locus. In preferred embodiments, the inventionprovides for an expanded targeting range for RNA guided genome editingnucleases wherein the T-rich PAMs of the Cpf1 family allow for targetingand editing of AT-rich genomes.

In certain embodiments, the CRISPR enzyme is engineered and can compriseone or more mutations that reduce or eliminate a nuclease activity. Theamino acid positions in the FnCpf1p RuvC domain include but are notlimited to D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A,E1028A, D1227A, D1255A and N1257A. Applicants have also identified aputative second nuclease domain which is most similar to PD-(D/E)XKnuclease superfamily and HincII endonuclease like. The point mutationsto be generated in this putative nuclease domain to substantially reducenuclease activity include but are not limited to N580A, N584A, T587A,W609A, D610A, K613A, E614A, D616A, K624A, D625A, K627A and Y629A. In apreferred embodiment, the mutation in the FnCpf1p RuvC domain is D917Aor E1006A, wherein the D917A or E1006A mutation completely inactivatesthe DNA cleavage activity of the FnCpf1 effector protein. In anotherembodiment, the mutation in the FnCpf1p RuvC domain is D1255A, whereinthe mutated FnCpf1 effector protein has significantly reducednucleolytic activity.

The amino acid positions in the AsCpf1p RuvC domain include but are notlimited to 908, 993, and 1263. In a preferred embodiment, the mutationin the AsCpf1p RuvC domain is D908A, E993A, and D1263A, wherein theD908A, E993A, and D1263A mutations completely inactivates the DNAcleavage activity of the AsCpf1 effector protein. The amino acidpositions in the LbCpf1p RuvC domain include but are not limited to 832,947 or 1180. In a preferred embodiment, the mutation in the LbCpf1p RuvCdomain is LbD832A, E925A, D947A or D1180A, wherein the LbD832A E925A,D947A or D1180A mutations completely inactivates the DNA cleavageactivity of the LbCpf1 effector protein.

Mutations can also be made at neighboring residues, e.g., at amino acidsnear those indicated above that participate in the nuclease acrivity. Insome embodiments, only the RuvC domain is inactivated, and in otherembodiments, another putative nuclease domain is inactivated, whereinthe effector protein complex functions as a nickase and cleaves only oneDNA strand. In a preferred embodiment, the other putative nucleasedomain is a HincII-like endonuclease domain. In some embodiments, twoFnCpf1, AsCpf1 or LbCpf1 variants (each a different nickase) are used toincrease specificity, two nickase variants are used to cleave DNA at atarget (where both nickases cleave a DNA strand, while minimizing oreliminating off-target modifications where only one DNA strand iscleaved and subsequently repaired). In preferred embodiments the Cpf1effector protein cleaves sequences associated with or at a target locusof interest as a homodimer comprising two Cpf1 effector proteinmolecules. In a preferred embodiment the homodimer may comprise two Cpf1effector protein molecules comprising a different mutation in theirrespective RuvC domains.

The invention contemplates methods of using two or more nickases, inparticular a dual or double nickase approach. In some aspects andembodiments, a single type FnCpf1, AsCpf1 or LbCpf1 nickase may bedelivered, for example a modified FnCpf1, AsCpf1 or LbCpf1 or a modifiedFnCpf1, AsCpf1 or LbCpf1 nickase as described herein. This results inthe target DNA being bound by two FnCpf1 nickases. In addition, it isalso envisaged that different orthologs may be used, e.g, an FnCpf1,AsCpf1 or LbCpf1 nickase on one strand (e.g., the coding strand) of theDNA and an ortholog on the non-coding or opposite DNA strand. Theortholog can be, but is not limited to, a Cas9 nickase such as a SaCas9nickase or a SpCas9 nickase. It may be advantageous to use two differentorthologs that require different PAMs and may also have different guiderequirements, thus allowing a greater deal of control for the user. Incertain embodiments, DNA cleavage will involve at least four types ofnickases, wherein each type is guided to a different sequence of targetDNA, wherein each pair introduces a first nick into one DNA strand andthe second introduces a nick into the second DNA strand. In suchmethods, at least two pairs of single stranded breaks are introducedinto the target DNA wherein upon introduction of first and second pairsof single-strand breaks, target sequences between the first and secondpairs of single-strand breaks are excised. In certain embodiments, oneor both of the orthologs is controllable, i.e. inducible.

In certain embodiments of the invention, the guide RNA or mature crRNAcomprises, consists essentially of, or consists of a direct repeatsequence and a guide sequence or spacer sequence. In certainembodiments, the guide RNA or mature crRNA comprises, consistsessentially of, or consists of a direct repeat sequence linked to aguide sequence or spacer sequence. In certain embodiments the guide RNAor mature crRNA comprises 19 nts of partial direct repeat followed by20-30 nt of guide sequence or spacer sequence, advantageously about 20nt, 23-25 nt or 24 nt. In certain embodiments, the effector protein is aFnCpf1, AsCpf1 or LbCpf1 effector protein and requires at least 16 nt ofguide sequence to achieve detectable DNA cleavage and a minimum of 17 ntof guide sequence to achieve efficient DNA cleavage in vitro. In certainembodiments, the direct repeat sequence is located upstream (i.e., 5′)from the guide sequence or spacer sequence. In a preferred embodimentthe seed sequence (i.e. the sequence essential critical for recognitionand/or hybridization to the sequence at the target locus) of the FnCpf1,AsCpf1 or LbCpf1 guide RNA is approximately within the first 5 nt on the5′ end of the guide sequence or spacer sequence.

In preferred embodiments of the invention, the mature crRNA comprises astem loop or an optimized stem loop structure or an optimized secondarystructure. In preferred embodiments the mature crRNA comprises a stemloop or an optimized stem loop structure in the direct repeat sequence,wherein the stem loop or optimized stem loop structure is important forcleavage activity. In certain embodiments, the mature crRNA preferablycomprises a single stem loop. In certain embodiments, the direct repeatsequence preferably comprises a single stem loop. In certainembodiments, the cleavage activity of the effector protein complex ismodified by introducing mutations that affect the stem loop RNA duplexstructure. In preferred embodiments, mutations which maintain the RNAduplex of the stem loop may be introduced, whereby the cleavage activityof the effector protein complex is maintained. In other preferredembodiments, mutations which disrupt the RNA duplex structure of thestem loop may be introduced, whereby the cleavage activity of theeffector protein complex is completely abolished.

The invention also provides for the nucleotide sequence encoding theeffector protein being codon optimized for expression in a eukaryote oreukaryotic cell in any of the herein described methods or compositions.In an embodiment of the invention, the codon optimized effector proteinis FnCpf1p, AsCpf1 or LbCpf1 and is codon optimized for operability in aeukaryotic cell or organism, e.g., such cell or organism as elsewhereherein mentioned, for instance, without limitation, a yeast cell, or amammalian cell or organism, including a mouse cell, a rat cell, and ahuman cell or non-human eukaryote organism, e.g., plant.

In certain embodiments of the invention, at least one nuclearlocalization signal (NLS) is attached to the nucleic acid sequencesencoding the Cpf1 effector proteins. In preferred embodiments at leastone or more C-terminal or N-terminal NLSs are attached (and hencenucleic acid molecule(s) coding for the Cpf1 effector protein caninclude coding for NLS(s) so that the expressed product has the NLS(s)attached or connected). In a preferred embodiment a C-terminal NLS isattached for optimal expression and nuclear targeting in eukaryoticcells, preferably human cells. In a preferred embodiment, the codonoptimized effector protein is FnCpf1p, AsCpf1 or LbCpf1 and the spacerlength of the guide RNA is from 15 to 35 nt. In certain embodiments, thespacer length of the guide RNA is at least 16 nucleotides, such as atleast 17 nucleotides. In certain embodiments, the spacer length is from15 to 17 nt, from 17 to 20 nt, from 20 to 24 nt, eg. 20, 21, 22, 23, or24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, from27-30 nt, from 30-35 nt, or 35 nt or longer. In certain embodiments ofthe invention, the codon optimized effector protein is FnCpf1p and thedirect repeat length of the guide RNA is at least 16 nucleotides. Incertain embodiments, the codon optimized effector protein is FnCpf1p andthe direct repeat length of the guide RNA is from 16 to 20 nt, e.g., 16,17, 18, 19, or 20 nucleotides. In certain preferred embodiments, thedirect repeat length of the guide RNA is 19 nucleotides.

The invention also encompasses methods for delivering multiple nucleicacid components, wherein each nucleic acid component is specific for adifferent target locus of interest thereby modifying multiple targetloci of interest. The nucleic acid component of the complex may compriseone or more protein-binding RNA aptamers. The one or more aptamers maybe capable of binding a bacteriophage coat protein. The bacteriophagecoat protein may be selected from the group comprising Qβ, F2, GA, fr,JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP,FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. Ina preferred embodiment the bacteriophage coat protein is MS2. Theinvention also provides for the nucleic acid component of the complexbeing 30 or more, 40 or more or 50 or more nucleotides in length.

The invention also encompasses the cells, components and/or systems ofthe present invention having trace amounts of cations present in thecells, components and/or systems. Advantageously, the cation ismagnesium, such as Mg²⁺. The cation may be present in a trace amount. Apreferred range may be about 1 mM to about 15 mM for the cation, whichis advantageously Mg²⁺. A preferred concentration may be about 1 mM forhuman based cells, components and/or systems and about 10 mM to about 15mM for bacteria based cells, components and/or systems. See, e.g.,Gasiunas et al., PNAS, published online Sep. 4, 2012,www.pnas.org/cgi/doi/10.1073/pnas.1208507109.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1B depict a new classification of CRISPR-Cas systems. Class 2includes multisubunit crRNA-effector complexes (Cascade) and Class 2includes Single-subunit crRNA-effector complexes (Cas9-like).

FIG. 2 provides a molecular organization of CRISPR-Cas.

FIGS. 3A-3D provide structures of Type I and III effector complexes:common architecture/common ancestry despite extensive sequencedivergence.

FIG. 4 shows CRISPR-Cas as a RNA recognition motif (RRM)-centeredsystem.

FIGS. 5A-5D show Cas1 phylogeny where recombination of adaptation andcrRNA-effector modules show a major aspect of CRISPR-Cas evolution.

FIG. 6 shows a CRISPR-Cas census, specifically a distribution ofCRISPR-Cas types/subtypes among archaea and bacteria

FIG. 7 depicts a pipeline for identifying Cas candidates.

FIGS. 8A-8D depict an organization of complete loci of Class 2 systems.

FIGS. 9A-9B depict C2c1 neighborhoods.

FIGS. 10A-10C depict a Cas1 tree.

FIGS. 11A-11B depict a domain organization of class 2 families.

FIGS. 12A-12B depict TnpB homology regions in Class 2 proteins (SEQ IDNOS 246-428, respectively, in order of appearance).

FIGS. 13A-13B depict C2c2 neighborhoods.

FIGS. 14A-14E depict HEPN RxxxxH motif in C2c2 family (SEQ ID NOS429-1032, respectively, in order of appearance).

FIG. 15 depicts C2C1: 1. Alicyclobacillus acidoterrestris ATCC 49025(SEQ ID NOS 1034-1037, respectively, in order of appearance).

FIG. 16 depicts C2C1: 4. Desulfonatronum thiodismutans strain MLF-1 (SEQID NOS 1038-1041, respectively, in order of appearance).

FIG. 17 depicts C2C1: 5. Opitutaceae bacterium TAV5 (SEQ ID NOS1042-1045, respectively, in order of appearance).

FIG. 18 depicts C2C1: 7. Bacillus thermoamylovorans strain B4166 (SEQ IDNOS 1046-1049, respectively, in order of appearance).

FIG. 19 depicts C2C1: 9. Bacillus sp. NSP2.1 (SEQ ID NOS 1050-1053,respectively, in order of appearance).

FIG. 20 depicts C2C2: 1. Lachnospiraceae bacterium MA2020 (SEQ ID NOS1054-1057, respectively, in order of appearance).

FIG. 21 depicts C2C2: 2. Lachnospiraceae bacterium NK4A179 (SEQ ID NOS1058-1064, respectively, in order of appearance).

FIG. 22 depicts C2C2: 3. [Clostridium] aminophilum DSM 10710 (SEQ ID NOS1065-1068, respectively, in order of appearance).

FIG. 23 depicts C2C2: 4. Lachnospiraceae bacterium NK4A144 (SEQ ID NOS1069 and 1070, respectively, in order of appearance).

FIG. 24 depicts C2C2: 5. Carnobacterium gallinarum DSM 4847 (SEQ ID NOS1071-1074, respectively, in order of appearance).

FIG. 25 depicts C2C2: 6. Carnobacterium gallinarum DSM 4847 (SEQ ID NOS1075-1081, respectively, in order of appearance).

FIG. 26 depicts C2C2: 7. Paludibacter propionicigenes WB4 (SEQ ID NO:1082).

FIG. 27 depicts C2C2: 8. Listeria seeligeri serovar 1/2b (SEQ ID NOS1083-1086, respectively, in order of appearance).

FIG. 28 depicts C2C2: 9. Listeria weihenstephanensis FSL R9-0317 (SEQ IDNO: 1087).

FIG. 29 depicts C2C2: 10. Listeria bacterium FSL M6-0635 (SEQ ID NOS1088 and 1091, respectively, in order of appearance).

FIG. 30 depicts C2C2: 11. Leptotrichia wadei F0279 (SEQ ID NO: 1092).

FIG. 31 depicts C2C2: 12. Leptotrichia wadei F0279 (SEQ ID NOS1093-1099, respectively, in order of appearance).

FIG. 32 depicts C2C2: 14. Leptotrichia shahii DSM 19757 (SEQ ID NOS1100-1103, respectively, in order of appearance).

FIG. 33 depicts C2C2: 15. Rhodobacter capsulatus SB 1003 (SEQ ID NOS1104 and 1105, respectively, in order of appearance).

FIG. 34 depicts C2C2: 16. Rhodobacter capsulatus R121 (SEQ ID NOS 1106and 1107, respectively, in order of appearance).

FIG. 35 depicts C2C2: 17. Rhodobacter capsulatus DE442 (SEQ ID NOS 1108and 1109, respectively, in order of appearance).

FIG. 36 depicts a tree of DRs

FIG. 37 depicts a tree of C2C2s

FIGS. 38A-38AH show the sequence alignment of Cas-Cpf1 orthologs (SEQ IDNOS 1033 and 1110-1166, respectively, in order of appearance).

FIGS. 39A-39C show the overview of Cpf1 loci alignment

FIGS. 40A-40X shows the PACYC184 FnCpf1 (PY001) vector construct (SEQ IDNO: 1167 and SEQ ID NOS 1168-1189, respectively, in order ofappearance).

FIGS. 41A-41I shows the sequence of humanized PaCpf1, with thenucleotide sequence as SEQ ID NO: 1190 and the protein sequence as SEQID NO: 1191.

FIG. 42 depicts a PAM challenge assay

FIG. 43 depicts a schematic of an endogenous FnCpf1 locus. pY0001 is apACY184 backbone (from NEB) with a partial FnCpf1 locus. The FnCpf1locus was PCR amplified in three pieces and cloned into Xba1 and Hind3cut pACYC184 using Gibson assembly. PY0001 contains the endogenousFnCpf1 locus from 255 bp of the acetyltransferase 3′ sequence to thefourth spacer sequence. Only spacer 1-3 are potentially active sincespace 4 is no longer flanked by direct repeats.

FIG. 44 depicts PAM libraries, which discloses SEQ ID NOS 1192-1195,respectively, in order of appearance. Both PAM libraries (left andright) are in pUC19. The complexity of left PAM library is 48˜65k andthe complexity of the right PAM library is 47˜16k. Both libraries wereprepared with a representation of >500.

FIG. 45A-45E depicts FnCpf1 PAM Screen Computational Analysis. Aftersequencing of the screen DNA, the regions corresponding to either theleft PAM or the right PAM were extracted. For each sample, the number ofPAMs present in the sequenced library were compared to the number ofexpected PAMs in the library (4{circumflex over ( )}8 for the leftlibrary, 4{circumflex over ( )}7 for the right). FIG. 44A depicts theleft library showed PAM depletion. To quantify this depletion, anenrichment ratio was calculated. For both conditions (control pACYC orFnCpf1 containing pACYC) the ratio was calculated for each PAM in thelibrary as

${ratio} = {{- \log_{2}}{\frac{{sample} + 0.01}{{{initial}\mspace{14mu}{library}} + 0.01}.}}$

Plotting the distribution shows little enrichment in the control sampleand enrichment in both bioreps. FIGS. 44B-44D depict PAM ratiodistributions. FIG. 44E shows PAMs above a ratio of 8 were collected,and the frequency distributions were plotted, revealing a 5′ YYN PAM.

FIG. 46 depicts RNAseq analysis of the Francisella tolerances Cpf1locus, which shows that the CRISPR locus is actively expressed. Inaddition to the Cpf1 and Cas genes, two small non-coding transcript arehighly transcribed, which might be the putative tracrRNAs. The CRISPRarray is also expressed. Both the putative tracrRNAs and CRISPR arrayare transcribed in the same direction as the Cpf1 and Cas genes. Hereall RNA transcripts identified through the RNAseq experiment are mappedagainst the locus. After further evaluation of the FnCpf1 locus,Applicants concluded that target DNA cleavage by a Cpf1 effector proteincomplex does not require a tracrRNA. Applicants determined that Cpf1effector protein complexes comprising only a Cpf1 effector protein and acrRNA (guide RNA comprising a direct repeat sequence and a guidesequence) were sufficient to cleave target DNA.

FIG. 47 depicts zooming into the Cpf1 CRISPR array. Many different shorttranscripts can be identified. In this plot, all identified RNAtranscripts are mapped against the Cpf1 locus.

FIG. 48 depicts identifying two putative tracrRNAs after selectingtranscripts that are less than 85 nucleotides long

FIG. 49 depicts zooming into putative tracrRNA 1 (SEQ ID NO: 1196) andthe CRISPR array

FIG. 50 depicts zooming into putative tracrRNA 2 which discloses SEQ IDNOS 1197-1203, respectively, in order of appearance.

FIG. 51 depicts putative crRNA sequences (repeat in blue, spacer inblack) (SEQ ID NOS 1205 and 1206, respectively, in order of appearance).

FIG. 52 shows a schematic of the assay to confirm the predicted FnCpf1PAM in vivo.

FIG. 53 shows FnCpf1 locus carrying cells and control cells transformedwith pUC19 encoding endogenous spacer 1 with 5′ TTN PAM.

FIG. 54 shows a schematic indicating putative tracrRNA sequencepositions in the FnCpf1 locus, the crRNA (SEQ ID NO: 1207) and the pUCprotospacer vector.

FIG. 55 is a gel showing the PCR fragment with TTa PAM and proto-spacer1sequence incubated in cell lysate.

FIG. 56 is a gel showing the pUC-spacer1 with different PAMs incubatedin cell lysate.

FIG. 57 is a gel showing the BasI digestion after incubation in celllysate.

FIG. 58 is a gel showing digestion results for three putative crRNAsequences (SEQ ID NO: 1208).

FIG. 59 is a gel showing testing of different lengths of spacer againsta piece of target DNA containing the target site:5′-TTAgagaagtcatttaataaggccactgttaaaa-3′ (SEQ ID NO: 1209). The resultsshow that crRNAs 1-7 mediated successful cleavage of the target DNA invitro with FnCpf1. crRNAs 8-13 did not facilitate cleavage of the targetDNA. SEQ ID NOS 1210-1248 are disclosed, respectively, in order ofappearance.

FIG. 60 is a schematic indicating the minimal FnCpf1 locus.

FIG. 61 is a schematic indicating the minimal Cpf1 guide (SEQ ID NO:1249).

FIG. 62A-62E depicts PaCpf1 PAM Screen Computational Analysis. Aftersequencing of the screen DNA, the regions corresponding to either theleft PAM or the right PAM were extracted. For each sample, the number ofPAMs present in the sequenced library were compared to the number ofexpected PAMs in the library (4{circumflex over ( )}7). (FIG. 62A) Theleft library showed very slight PAM depletion. To quantify thisdepletion, an enrichment ratio was calculated. For both conditions(control pACYC or PaCpf1 containing pACYC) the ratio was calculated foreach PAM in the library as

${ratio} = {{- \log_{2}}\frac{{sample} + 0.01}{{{initial}\mspace{14mu}{library}} + 0.01}}$

Plotting the distribution shows little enrichment in the control sampleand enrichment in both bioreps. FIGS. 62B-62D depict PAM ratiodistributions. FIG. 62E shows all PAMs above a ratio of 4.5 werecollected, and the frequency distributions were plotted, revealing a 5′TTTV PAM, where V is A or C or G.

FIG. 63 shows a vector map of the human codon optimized PaCpf1 sequencedepicted as CBh-NLS-huPaCpf1-NLS-3xHA-pA.

FIGS. 64A-64B show a phylogenetic tree of 51 Cpf1 loci in differentbacteria. Highlighted boxes indicate Gene Reference #s: 1-17.Boxed/numbered orthologs were tested for in vitro cleavage activity withpredicted mature crRNA; orthologs with boxes around their numbers showedactivity in the in vitro assay.

FIGS. 65A-65H show the details of the human codon optimized sequence forLachnospiraceae bacterium MC2017 1 Cpf1 having a gene length of 3849 nts(Ref #3 in FIG. 64 ). FIG. 65A: Codon Adaptation Index (CAI). Thedistribution of codon usage frequency along the length of the genesequence. A CAI of 1.0 is considered to be perfect in the desiredexpression organism, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. FIG. 65B: Frequency of Optimal Codons (FOP).The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression organism. FIG. 65C: GCContent Adjustment. The ideal percentage range of GC content is between30-70%. Peaks of % GC content in a 60 bp window have been removed. FIG.65D: Restriction Enzymes and CIS-Acting Elements. FIG. 65E: RemoveRepeat Sequences. FIG. 65F-G: Optimized Sequence (Optimized SequenceLength: 3849, GC % 54.70) (SEQ ID NO: 1250). FIG. 65H: Protein Sequence(SEQ ID NO: 1251).

FIGS. 66A-66H show the details of the human codon optimized sequence forButyrivibrio proteoclasticus Cpf1 having a gene length of 3873 nts (Ref#4 in FIG. 64 ). FIG. 66A: Codon Adaptation Index (CAI). Thedistribution of codon usage frequency along the length of the genesequence. A CAI of 1.0 is considered to be perfect in the desiredexpression organism, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. FIG. 66B: Frequency of Optimal Codons (FOP).The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression organism. FIG. 66C: GCContent Adjustment. The ideal percentage range of GC content is between30-70%. Peaks of % GC content in a 60 bp window have been removed. FIG.66D: Restriction Enzymes and CIS-Acting Elements. FIG. 66E: RemoveRepeat Sequences. FIG. 66F-G: Optimized Sequence (Optimized SequenceLength: 3873, GC % 54.05) (SEQ ID NO: 1252). FIG. 66H: Protein Sequence(SEQ ID NO: 1253).

FIGS. 67A-67H show the details of the human codon optimized sequence forPeregrinibacteria bacterium GW2011_GWA2_33_10 Cpf1 having a gene lengthof 4581 nts (Ref #5 in FIG. 64 ). FIG. 67A: Codon Adaptation Index(CAI). The distribution of codon usage frequency along the length of thegene sequence. A CAI of 1.0 is considered to be perfect in the desiredexpression organism, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. FIG. 67B: Frequency of Optimal Codons (FOP).The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression organism. FIG. 67C: GCContent Adjustment. The ideal percentage range of GC content is between30-70%. Peaks of % GC content in a 60 bp window have been removed. FIG.67D: Restriction Enzymes and CIS-Acting Elements. FIG. 67E: RemoveRepeat Sequences. FIG. 67F-G: Optimized Sequence (Optimized SequenceLength: 4581, GC % 50.81) (SEQ ID NO: 1254). FIG. 67H: Protein Sequence(SEQ ID NO: 1255).

FIGS. 68A-68H show the details of the human codon optimized sequence forParcubacteria bacterium GW2011_GWC2_44_17 Cpf1 having a gene length of4206 nts (Ref #6 in FIG. 64 ). FIG. 68A: Codon Adaptation Index (CAI).The distribution of codon usage frequency along the length of the genesequence. A CAI of 1.0 is considered to be perfect in the desiredexpression organism, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. FIG. 68B: Frequency of Optimal Codons (FOP).The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression organism. FIG. 68C: GCContent Adjustment. The ideal percentage range of GC content is between30-70%. Peaks of % GC content in a 60 bp window have been removed. FIG.68D: Restriction Enzymes and CIS-Acting Elements. FIG. 68E: RemoveRepeat Sequences. FIG. 68F-G: Optimized Sequence (Optimized SequenceLength: 4206, GC % 52.17) (SEQ ID NO: 1256). FIG. 68H: Protein Sequence(SEQ ID NO: 1257).

FIGS. 69A-69H show the details of the human codon optimized sequence forSmithella sp. SCADC Cpf1 having a gene length of 3900 nts (Ref #7 inFIG. 64 ). FIG. 69A: Codon Adaptation Index (CAI). The distribution ofcodon usage frequency along the length of the gene sequence. A CAI of1.0 is considered to be perfect in the desired expression organism, anda CAI of >0.8 is regarded as good, in terms of high gene expressionlevel. FIG. 69B: Frequency of Optimal Codons (FOP). The percentagedistribution of codons in computed codon quality groups. The value of100 is set for the codon with the highest usage frequency for a givenamino acid in the desired expression organism. FIG. 69C: GC ContentAdjustment. The ideal percentage range of GC content is between 30-70%.Peaks of % GC content in a 60 bp window have been removed. FIG. 69D:Restriction Enzymes and CIS-Acting Elements. FIG. 69E: Remove RepeatSequences. FIG. 69F-G: Optimized Sequence (Optimized Sequence Length:3900, GC % 51.56) (SEQ ID NO: 1258). FIG. 69H: Protein Sequence (SEQ IDNO: 1259).

FIGS. 70A-70H show the details of the human codon optimized sequence forAcidaminococcus sp. BV3L6 Cpf1 having a gene length of 4071 nts (Ref #8in FIG. 64 ). FIG. 70A: Codon Adaptation Index (CAI). The distributionof codon usage frequency along the length of the gene sequence. A CAI of1.0 is considered to be perfect in the desired expression organism, anda CAI of >0.8 is regarded as good, in terms of high gene expressionlevel. FIG. 70B: Frequency of Optimal Codons (FOP). The percentagedistribution of codons in computed codon quality groups. The value of100 is set for the codon with the highest usage frequency for a givenamino acid in the desired expression organism. FIG. 70C: GC ContentAdjustment. The ideal percentage range of GC content is between 30-70%.Peaks of % GC content in a 60 bp window have been removed. FIG. 70D:Restriction Enzymes and CIS-Acting Elements. FIG. 70E: Remove RepeatSequences. FIG. 70F-G: Optimized Sequence (Optimized Sequence Length:4071, GC % 54.89) (SEQ ID NO: 1260). FIG. 70H: Protein Sequence (SEQ IDNO: 1261).

FIGS. 71A-71H show the details of the human codon optimized sequence forLachnospiraceae bacterium MA2020 Cpf1 having a gene length of 3768 nts(Ref #9 in FIG. 64 ). FIG. 71A: Codon Adaptation Index (CAI). Thedistribution of codon usage frequency along the length of the genesequence. A CAI of 1.0 is considered to be perfect in the desiredexpression organism, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. FIG. 71B: Frequency of Optimal Codons (FOP).The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression organism. FIG. 71C: GCContent Adjustment. The ideal percentage range of GC content is between30-70%. Peaks of % GC content in a 60 bp window have been removed. FIG.71D: Restriction Enzymes and CIS-Acting Elements. FIG. 71E: RemoveRepeat Sequences. FIG. 71F-G: Optimized Sequence (Optimized SequenceLength: 3768, GC % 51.53) (SEQ ID NO: 1262). FIG. 71H: Protein Sequence(SEQ ID NO: 1263).

FIGS. 72A-72H show the details of the human codon optimized sequence forCandidatus Methanoplasma termitum Cpf1 having a gene length of 3864 nts(Ref #10 in FIG. 64 ). FIG. 72A: Codon Adaptation Index (CAI). Thedistribution of codon usage frequency along the length of the genesequence. A CAI of 1.0 is considered to be perfect in the desiredexpression organism, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. FIG. 72B: Frequency of Optimal Codons (FOP).The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression organism. FIG. 72C: GCContent Adjustment. The ideal percentage range of GC content is between30-70%. Peaks of % GC content in a 60 bp window have been removed. FIG.72D: Restriction Enzymes and CIS-Acting Elements. FIG. 72E: RemoveRepeat Sequences. FIG. 72F-G: Optimized Sequence (Optimized SequenceLength: 3864, GC % 52.67) (SEQ ID NO: 1264). FIG. 72H: Protein Sequence(SEQ ID NO: 1265).

FIGS. 73A-73H show the details of the human codon optimized sequence forEubacterium eligens Cpf1 having a gene length of 3996 nts (Ref #11 inFIG. 64 ). FIG. 73A: Codon Adaptation Index (CAI). The distribution ofcodon usage frequency along the length of the gene sequence. A CAI of1.0 is considered to be perfect in the desired expression organism, anda CAI of >0.8 is regarded as good, in terms of high gene expressionlevel. FIG. 73B: Frequency of Optimal Codons (FOP). The percentagedistribution of codons in computed codon quality groups. The value of100 is set for the codon with the highest usage frequency for a givenamino acid in the desired expression organism. FIG. 73C: GC ContentAdjustment. The ideal percentage range of GC content is between 30-70%.Peaks of % GC content in a 60 bp window have been removed. FIG. 73D:Restriction Enzymes and CIS-Acting Elements. FIG. 73E: Remove RepeatSequences. FIG. 73F-G: Optimized Sequence (Optimized Sequence Length:3996, GC % 50.52) (SEQ ID NO: 1266). FIG. 73H: Protein Sequence (SEQ IDNO: 1267).

FIGS. 74A-74H show the details of the human codon optimized sequence forMoraxella bovoculi 237 Cpf1 having a gene length of 4269 nts (Ref #12 inFIG. 64 ). FIG. 74A: Codon Adaptation Index (CAI). The distribution ofcodon usage frequency along the length of the gene sequence. A CAI of1.0 is considered to be perfect in the desired expression organism, anda CAI of >0.8 is regarded as good, in terms of high gene expressionlevel. FIG. 74B: Frequency of Optimal Codons (FOP). The percentagedistribution of codons in computed codon quality groups. The value of100 is set for the codon with the highest usage frequency for a givenamino acid in the desired expression organism. FIG. 74C: GC ContentAdjustment. The ideal percentage range of GC content is between 30-70%.Peaks of % GC content in a 60 bp window have been removed. FIG. 74D:Restriction Enzymes and CIS-Acting Elements. FIG. 74E: Remove RepeatSequences. FIG. 74F-G: Optimized Sequence (Optimized Sequence Length:4269, GC % 53.58) (SEQ ID NO: 1268). FIG. 74H: Protein Sequence (SEQ IDNO: 1269).

FIGS. 75A-75H show the details of the human codon optimized sequence forLeptospira inadai Cpf1 having a gene length of 3939 nts (Ref #13 in FIG.64 ). FIG. 75A: Codon Adaptation Index (CAI). The distribution of codonusage frequency along the length of the gene sequence. A CAI of 1.0 isconsidered to be perfect in the desired expression organism, and a CAIof >0.8 is regarded as good, in terms of high gene expression level.FIG. 75B: Frequency of Optimal Codons (FOP). The percentage distributionof codons in computed codon quality groups. The value of 100 is set forthe codon with the highest usage frequency for a given amino acid in thedesired expression organism. FIG. 75C: GC Content Adjustment. The idealpercentage range of GC content is between 30-70%. Peaks of % GC contentin a 60 bp window have been removed. FIG. 75D: Restriction Enzymes andCIS-Acting Elements. FIG. 75E: Remove Repeat Sequences. FIG. 75F-G:Optimized Sequence (Optimized Sequence Length: 3939, GC % 51.30) (SEQ IDNO: 1270). FIG. 75H: Protein Sequence (SEQ ID NO: 1271).

FIGS. 76A-76H show the details of the human codon optimized sequence forLachnospiraceae bacterium ND2006 Cpf1 having a gene length of 3834 nts(Ref #14 in FIG. 64 ). FIG. 76A: Codon Adaptation Index (CAI). Thedistribution of codon usage frequency along the length of the genesequence. A CAI of 1.0 is considered to be perfect in the desiredexpression organism, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. FIG. 76B: Frequency of Optimal Codons (FOP).The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression organism. FIG. 76C: GCContent Adjustment. The ideal percentage range of GC content is between30-70%. Peaks of % GC content in a 60 bp window have been removed. FIG.76D: Restriction Enzymes and CIS-Acting Elements. FIG. 76E: RemoveRepeat Sequences. FIG. 76F-G: Optimized Sequence (Optimized SequenceLength: 3834, GC % 51.06) (SEQ ID NO: 1272). FIG. 76H: Protein Sequence(SEQ ID NO: 1273).

FIGS. 77A-77H show the details of the human codon optimized sequence forPorphyromonas crevioricanis 3 Cpf1 having a gene length of 3930 nts (Ref#15 in FIG. 64 ). FIG. 77A: Codon Adaptation Index (CAI). Thedistribution of codon usage frequency along the length of the genesequence. A CAI of 1.0 is considered to be perfect in the desiredexpression organism, and a CAI of >0.8 is regarded as good, in terms ofhigh gene expression level. FIG. 77B: Frequency of Optimal Codons (FOP).The percentage distribution of codons in computed codon quality groups.The value of 100 is set for the codon with the highest usage frequencyfor a given amino acid in the desired expression organism. FIG. 77C: GCContent Adjustment. The ideal percentage range of GC content is between30-70%. Peaks of % GC content in a 60 bp window have been removed. FIG.77D: Restriction Enzymes and CIS-Acting Elements. FIG. 77E: RemoveRepeat Sequences. FIG. 77F-G: Optimized Sequence (Optimized SequenceLength: 3930, GC % 54.42) (SEQ ID NO: 1274). FIG. 77H: Protein Sequence(SEQ ID NO: 1275).

FIGS. 78A-78H show the details of the human codon optimized sequence forPrevotella disiens Cpf1 having a gene length of 4119 nts (Ref #16 inFIG. 64 ). FIG. 78A: Codon Adaptation Index (CAI). The distribution ofcodon usage frequency along the length of the gene sequence. A CAI of1.0 is considered to be perfect in the desired expression organism, anda CAI of >0.8 is regarded as good, in terms of high gene expressionlevel. FIG. 78B: Frequency of Optimal Codons (FOP). The percentagedistribution of codons in computed codon quality groups. The value of100 is set for the codon with the highest usage frequency for a givenamino acid in the desired expression organism. FIG. 78C: GC ContentAdjustment. The ideal percentage range of GC content is between 30-70%.Peaks of % GC content in a 60 bp window have been removed. FIG. 78D:Restriction Enzymes and CIS-Acting Elements. FIG. 78E: Remove RepeatSequences. FIG. 78F-G: Optimized Sequence (Optimized Sequence Length:4119, GC % 51.88) (SEQ ID NO: 1276). FIG. 78H: Protein Sequence (SEQ IDNO: 1277).

FIGS. 79A-79H shows the details of the human codon optimized sequencefor Porphyromonas macacae Cpf1 having a gene length of 3888 nts (Ref #17in FIG. 64 ). FIG. 79A: Codon Adaptation Index (CAI). The distributionof codon usage frequency along the length of the gene sequence. A CAI of1.0 is considered to be perfect in the desired expression organism, anda CAI of >0.8 is regarded as good, in terms of high gene expressionlevel. FIG. 79B: Frequency of Optimal Codons (FOP). The percentagedistribution of codons in computed codon quality groups. The value of100 is set for the codon with the highest usage frequency for a givenamino acid in the desired expression organism. FIG. 79C: GC ContentAdjustment. The ideal percentage range of GC content is between 30-70%.Peaks of % GC content in a 60 bp window have been removed. FIG. 79D:Restriction Enzymes and CIS-Acting Elements. FIG. 79E: Remove RepeatSequences. FIG. 79F-G: Optimized Sequence (Optimized Sequence Length:3888, GC % 53.26) (SEQ ID NO: 1278). FIG. 79H: Protein Sequence (SEQ IDNO: 1279).

FIG. 80A-80I shows direct repeat (DR) sequences for each ortholog (referto numbering Ref #3-17 in FIG. 64 ) and their predicted fold structure.SEQ ID NOS 1280-1313, respectively, are disclosed in order ofappearance.

FIG. 81 shows cleavage of a PCR amplicon of the human Emx1 locus. SEQ IDNOS 1314-1318, respectively, are disclosed in order of appearance.

FIG. 82A-82B shows the effect of truncation in 5′ DR on cleavageActivity. FIG. 82A shows a gel in which cleavage results with 5 DRtruncations is indicated. FIG. 82B shows a diagram in which crDNAdeltaDR5 disrupted the stem loop at the 5′ end. This indicates that thestemloop at the 5′ end is essential for cleavage activity. SEQ ID NOS1319-1324, respectively, are disclosed in order of appearance.

FIG. 83 shows the effect of crRNA-DNA target mismatch on cleavageefficiency. SEQ ID NOS 1325-1335, respectively, are disclosed in orderof appearance.

FIG. 84 shows the cleavage of DNA using purified Francisella andPrevotella Cpf1. SEQ ID NO: 1336 is disclosed.

FIG. 85A-85B show diagrams of DR secondary structures. FIG. 85A shows aFnCpf1 DR secondary structure (SEQ ID NO: 1337) (stem loop highlighted).FIG. 85B shows a PaCpf1 DR secondary structure (SEQ ID NO: 1338) (stemloop highlighted, identical except for a single base difference in theloop region).

FIG. 86 shows a further depiction of the RNAseq analysis of the FnCp1locus.

FIG. 87A-87B show schematics of mature crRNA sequences. FIG. 87A shows amature crRNA sequences for FnCpf1. FIG. 87B shows a mature crRNAsequences for PaCpf1. SEQ ID NOS 1339-1342, respectively, are disclosedin order of appearance.

FIG. 88 shows cleavage of DNA using human codon optimized Francisellanovicida FnCpf1. The top band corresponds to un-cleaved full lengthfragment (606 bp). Expected cleavage product sizes of ˜345 bp and ˜261bp are indicated by triangles.

FIG. 89 shows in vitro ortholog assay demonstrating cleavage by Cpf1orthologs.

FIGS. 90A-90C show computationally derived PAMs from the in vitrocutting assay.

FIG. 91 shows Cpf1 cutting in a staggered fashion with 5′ overhangs. SEQID NOS 1343-1345, respectively, are disclosed in order of appearance.

FIG. 92 shows effect of spacer length on cutting. SEQ ID NOS 1346-1352,respectively, are disclosed in order of appearance.

FIG. 93 shows SURVEYOR data for FnCpf1 mediated indels in HEK293T cells.

FIGS. 94A-94F show the processing of transcripts when sections of theFnCpf1 locus are deleted as compared to the processing of transcripts ina wild type FnCpf1 locus. FIGS. 95B, 95D and 95F zoom in on theprocessed spacer. SEQ ID NOS 1353-1401, respectively, are disclosed inorder of appearance.

FIGS. 95A-95E show the Francisella tularensis subsp. novicida U112 Cpf1CRISPR locus provides immunity against transformation of plasmidscontaining protospacers flanked by a 5′-TTN PAM. FIG. 95A show theorganization of two CRISPR loci found in Francisella tularensis subsp.novicida U112 (NC_008601). The domain organization of FnCas9 and FnCpf1are compared. FIG. 95B provide a schematic illustration of the plasmiddepletion assay for discovering the PAM position and identity. CompetentE. coli harboring either the heterologous FnCpf1 locus plasmid (pFnCpf1)or the empty vector control were transformed with a library of plasmidscontaining the matching protospacer flanked by randomized 5′ or 3′ PAMsequences and selected with antibiotic to deplete plasmids carryingsuccessfully-targeted PAM. Plasmids from surviving colonies wereextracted and sequenced to determine depleted PAM sequences. FIGS.95C-95D show sequence logos for the FnCpf1 PAM as determined by theplasmid depletion assay. Letter height at position is determined byinformation content; error bars show 95% Bayesian confidence interval.FIG. 95E shows E. coli harboring pFnCpf1 demonstrate robust interferenceagainst plasmids carrying 5′-TTN PAMs (n=3, error bars representmean±S.E.M.).

FIGS. 96A-96C shows heterologous expression of FnCpf1 and CRISPR arrayin E. coli is sufficient to mediate plasmid DNA interference and crRNAmaturation. Small RNA-seq of Francisella tularensis subsp. novicida U112(FIG. 96A) reveals transcription and processing of the FnCpf1 CRISPRarray. The mature crRNA begins with a 19 nt partial direct repeatfollowed by 23-25 nt of spacer sequence. Small RNA-seq of E. colitransformed with a plasmid carrying synthetic promoter-driven FnCpf1 andCRISPR array (FIG. 96B) shows crRNA processing independent of Cas genesand other sequence elements in the FnCpf1 locus. FIG. 96C depicts E.coli harboring different truncations of the FnCpf1 CRISPR locus andshows that only FnCpf1 and the CRISPR array are required for plasmid DNAinterference (n=3, error bars show mean±S.E.M.). SEQ ID NO: 1580 isdisclosed.

FIGS. 97A-97E shows FnCpf1 is targeted by crRNA to cleave DNA in vitro.FIG. 97A is a schematic of the FnCpf1 crRNA-DNA targeting complex.Cleavage sites are indicated by red arrows (SEQ ID NOS 1402 and 1403,respectively, disclosed in order of appearance). FnCpf1 and crRNA alonemediated RNA-guided cleavage of target DNA in a crRNA- andMg²⁺-dependent manner (FIG. 97B). FIG. 97C shows FnCpf1 cleaves bothlinear and supercoiled DNA. FIG. 97D shows Sanger sequencing traces fromFnCpf1-digested target show staggered overhangs (SEQ ID NOS 1404 and1406, respectively, disclosed in order of appearance). The non-templatedaddition of an additional adenine, denoted as N, is an artifact of thepolymerase used in sequencing. Reverse primer read represented asreverse complement to aid visualization. FIG. 97E shows cleavage isdependent on base-pairing at the 5′ PAM. FnCpf1 can only recognize thePAM in correctly Watson-Crick paired DNA.

FIGS. 98A-98B shows catalytic residues in the C-terminal RuvC domain ofFnCpf1 are necessary for DNA cleavage. FIG. 98A shows the domainstructure of FnCpf1 with RuvC catalytic residues highlighted. Thecatalytic residues were identified based on sequence homology to Thermusthermophilus RuvC (PDB ID: 4EP5). FIG. 98B depicts a native TBE PAGE gelshowing that mutation of the RuvC catalytic residues of FnCpf1 (D917Aand E1006A) and mutation of the RuvC (D10A) catalytic residue of SpCas9prevents double stranded DNA cleavage. Denaturing TBE-Urea PAGE gelshowing that mutation of the RuvC catalytic residues of FnCpf1 (D917Aand E1006A) prevents DNA nicking activity, whereas mutation of the RuvC(D10A) catalytic residue of SpCas9 results in nicking of the targetsite.

FIGS. 99A-99E shows crRNA requirements for FnCpf1 nuclease activity invitro. FIG. 99A shows the effect of spacer length on FnCpf1 cleavageactivity. FIG. 99B shows the effect of crRNA-target DNA mismatch onFnCpf1 cleavage activity. FIG. 99C demonstrates the effect of directrepeat length on FnCpf1 cleavage activity. FIG. 99D shows FnCpf1cleavage activity depends on secondary structure in the stem of thedirect repeat RNA structure. FIG. 99E shows FnCpf1 cleavage activity isunaffected by loop mutations but is sensitive to mutation in the 3′-mostbase of the direct repeat. SEQ ID NOS 1407-1433, respectively, disclosedin order of appearance.

FIGS. 100A-100F provides an analysis of Cpf1-family protein diversityand function. FIGS. 100A-100B show a phylogenetic comparison of 16 Cpf1orthologs selected for functional analysis. Conserved sequences areshown in dark gray. The RuvC domain, bridge helix, and zinc finger arehighlighted. FIG. 100C shows an alignment of direct repeats from the 16Cpf1-family proteins. Sequences that are removed post crRNA maturationare colored gray. Non-conserved bases are colored red. The stem duplexis highlighted in gray. FIG. 100D depicts RNAfold (Lorenz et al., 2011)prediction of the direct repeat sequence in the mature crRNA.Predictions for FnCpf1 along with three less-conserved orthologs shown.FIG. 100E shows ortholog crRNAs with similar direct repeat sequences areable to function with FnCpf1 to mediate target DNA cleavage. FIG. 100Fshows PAM sequences for 8 Cpf1-family proteins identified using in vitrocleavage of a plasmid library containing randomized PAMs flanking theprotospacer. SEQ ID NOS 1434-1453, respectively, disclosed in order ofappearance.

FIGS. 101A-101E shows Cpf1 mediates robust genome editing in human celllines. FIG. 101A is a schemative showing expression of individualCpf1-family proteins in HEK 293FT cells using CMV-driven expressionvectors. The corresponding crRNA is expressed using a PCR fragmentcontaining a U6 promoter fused to the crRNA sequence. Transfected cellswere analyzed using either Surveyor nuclease assay or targeted deepsequencing. FIG. 101B (top) depicts the sequence of DNMT1-targetingcrRNA 3, and sequencing reads (bottom) show representative indels. IG.101B discloses SEQ ID NOS 1454-1465, respectively, in order ofappearance. FIG. 101C provides a comparison of in vitro and in vivocleavage activity. The DNMT1 target region was PCR amplified and thegenomic fragment was used to test Cpf1-mediated cleavage. All 8Cpf1-family proteins showed DNA cleavage in vitro (top). Candidates7—AsCpf1 and 13—Lb3Cpf1 facilitated robust indel formation in humancells (bottom). FIG. 101D shows Cpf1 and SpCas9 target sequences in thehuman DNMT1 locus (SEQ ID NOS 1466-1473, respectively, disclosed inorder of appearance). FIG. 101E provides a comparison of Cpf1 and SpCas9genome editing efficiency. Target sites correspond to sequences shown inFIG. 101D.

FIGS. 102A-102D shows an in vivo plasmid depletion assay for identifyingFnCpf1 PAM. (See also FIG. 95 ). FIG. 102A: Transformation of E. coliharboring pFnCpf1 with a library of plasmids carrying randomized 5′ PAMsequences. A subset of plasmids were depleted. Plot shows depletionlevels in ranked order. Depletion is measured as the negative log₂ foldratio of normalized abundance compared pACYC184 E. coli controls. PAMsabove a threshold of 3.5 are used to generate sequence logos. FIG. 102B:Transformation of E. coli harboring pFnCpf1 with a library of plasmidscarrying randomized 3′ PAM sequences. A subset of plasmids weredepleted. Plot shows depletion levels in ranked order. Depletion ismeasured as the negative log₂ fold ratio of normalized abundancecompared pACYC184 E. coli controls and PAMs above a threshold of 3.5 areused to generate sequence logos. FIG. 102C: Input library of plasmidscarrying randomized 5′ PAM sequences. Plot shows depletion levels inranked order. Depletion is measured as the negative log₂ fold ratio ofnormalized abundance compared pACYC184 E. coli controls. PAMs above athreshold of 3.5 are used to generate sequence logos. FIG. 102D: Thenumber of unique PAMs passing significance threshold for pairwisecombinations of bases at the 2 and 3 positions of the 5′ PAM.

FIGS. 103A-103D shows FnCpf1 Protein Purification. (See also FIG. 97 ).FIG. 103A depicts a Coomassie blue stained acrylamide gel of FnCpf1showing stepwise purification. A band just above 160 kD eluted from theNi-NTA column, consistent with the size of a MBP-FnCpf1 fusion (189.7kD). Upon addition of TEV protease a lower molecular weight bandappeared, consistent with the size of 147 kD free FnCpf1. FIG. 103B:Size exclusion gel filtration of fnCpf1. FnCpf1 eluted at a sizeapproximately 300 kD (62.65 mL), suggesting Cpf1 may exist in solutionas a dimer. FIG. 103C shows protein standards used to calibrate theSuperdex 200 column. BDex=Blue Dextran (void volume), Ald=Aldolase (158kD), Ov=Ovalbumin (44 kD), RibA=Ribonuclease A (13.7 kD), Apr=Aprotinin(6.5 kD). FIG. 103D: Calibration curve of the Superdex 200 column. K_(a)is calculated as (elution volume−void volume)/(geometric columnvolume−void volume). Standards were plotted and fit to a logarithmiccurve.

FIGS. 104A-104E shows cleavage patterns of FnCpf1. (See also FIG. 97 ).Sanger sequencing traces from FnCpf1-digested DNA targets show staggeredoverhangs. The non-templated addition of an additional adenine, denotedas N, is an artifact of the polymerase used in sequencing. Sanger tracesare shown for different TTN PAMs with protospacer 1 (FIG. 104A),protospacer 2 (FIG. 104B), and protospacer 3 (FIG. 104C) and targetsDNMT1 and EMX1 (FIG. 104D). The (−) strand sequence isreverse-complemented to show the top strand sequence. Cleavage sites areindicated by red triangles. Smaller triangles indicate putativealternative cleavage sites. FIG. 104E shows the effect of PAM-distalcrRNA-target DNA mismatch on FnCpf1 cleavage activity. SEQ ID NOS1474-1494, respectively, disclosed in order of appearance.

FIGS. 105A-105B shows an amino acid sequence alignment of FnCpf1 (SEQ IDNO: 1495), AsCpf1 (SEQ ID NO: 1496), and LbCpf1 (SEQ ID NO: 1497). (Seealso FIG. 100 ). Residues that are conserved are highlighted with a redbackground and conserved mutations are highlighted with an outline andred font. Secondary structure prediction is highlighted above (FnCpf1)and below (LbCpf1) the alignment. Alpha helices are shown as a curlysymbol and beta strands are shown as dashes. Protein domains identifiedin FIG. 95A are also highlighted.

FIGS. 106A-106D provides maps bacterial genomic loci corresponding tothe 16 Cpf1-family proteins selected for mammalian experimentation. (Seealso FIG. 100 ). FIGS. 106A-106D disclose SEQ ID NOS 1498-1513,respectively, in order of appearance.

FIGS. 107A-107E shows in vitro characterization of Cpf1-family proteins.FIG. 107A is a schematic for in vitro PAM screen using Cpf1-familyproteins. A library of plasmids bearing randomized 5′ PAM sequences werecleaved by individual Cpf1-family proteins and their correspondingcrRNAs. Uncleaved plasmid DNA was purified and sequenced to identifyspecific PAM motifs that were depleted. FIG. 107B indicates the numberof unique sequences passing significance threshold for pairwisecombinations of bases at the 2 and 3 positions of the 5′ PAM for7—AsCpf1. FIG. 107C indicates the number of unique PAMs passingsignificance threshold for triple combinations of bases at the 2, 3, and4 positions of the 5′ PAM for 13—LbCpf1. FIGS. 107D-107E E and F showSanger sequencing traces from 7—AsCpf1-digested target (FIG. 107E) and13—LbCpf1-digested target (FIG. 107F) and show staggered overhangs. Thenon-templated addition of an additional adenine, denoted as N, is anartifact of the polymerase used in sequencing. Cleavage sites areindicated by red triangles. Smaller triangles indicate putativealternative cleavage sites. FIG. 107D-E discloses SEQ ID NOS 1514-1519,respectively, in order of appearance.

FIGS. 108A-108F indicates human cell genome editing efficiency atadditional loci. Surveyor gels show quantification of indel efficiencyachieved by each Cpf1-family protein at DNMT1 target sites 1 (FIG.108A), 2 (FIG. 108B), and 4 (FIG. 108C). FIGS. 108A-108C indicate humancell genome editing efficiency at additional loci and Sanger sequencingof cleaved of DNMT target sites. Surveyor gels show quantification ofindel efficiency achieved by each Cpf1-family protein at EMX1 targetsites 1 (FIG. 108D) and 2 (FIG. 108E). Indel distributions for AsCpf1and LbCpf1 and DNMT1 target sites 2, 3, and 4 (FIG. 108F). Cyan barsrepresent total indel coverage; blue bars represent distribution of 3′ends of indels. For each target, PAM sequence is in red and targetsequence is in light blue.

FIGS. 109A-109C depicts a computational analysis of the primarystructure of Cpf1 nucleases reveals three distinct regions. First aC-terminal RuvC like domain, which is the only functional characterizeddomain. Second a N-terminal alpha-helical region and thirst a mixedalpha and beta region, located between the RuvC like domain and thealpha-helical region.

FIGS. 110A-110E depict an AsCpf1 Rad50 alignment (PDB 4W9M). SEQ ID NOS1520 and 1521, respectively, disclosed in order of appearance. FIG. 110Cdepicts an AsCpf1 RuvC alignment (PDB 4LD0). SEQ ID NOS 1522 and 1523,respectively, disclosed in order of appearance. FIGS. 110D-110E depictsan alignment of AsCpf1 and FnCpf1 which identifies Rad50 domain inFnCpf1. SEQ ID NOS 1524 and 1525, respectively, disclosed in order ofappearance.

FIG. 111 depicts a structure of Rad50 (4W9M) in complex with DNA. DNAinteracting residues are highlighted (in red).

FIG. 112 depicts a structure of RuvC (4LD0) in complex with holidayjunction. DNA interacting residues are highlighted in red.

FIG. 113 depicts a blast of AsCpf1 aligns to a region of the sitespecific recombinase XerD. An active site regions of XerD is LYWTGMR(SEQ ID NO: 1) with R being a catalytic residue. SEQ ID NOS 1526-1527,respectively, disclosed in order of appearance.

FIG. 114 depicts a region is conserved in Cpf1 orthologs (Yellow box)and although the R is not conserved, a highly conserved aspartic acid(orange box) is just C-terminal of this region and a nearby conservedregion (blue box) with an absolutely conserved arginine. The asparticacid is D732 in LbCpf1. SEQ ID NOS 1204 and 1528-1579, respectively,disclosed in order of appearance.

FIG. 115A shows an experiment where 150,000 HEK293T cells were platedper 24-well 24 h before transfection. Cells were transfected with 400 nghuAsCpf1 plasmid and 100 ng of tandem guide plasmid comprising one guidesequence directed to GRIN28 and one directed to EMX1 placed in tandembehind the U6 promoter, using Lipofectamin2000. Cells were harvested 72h after transfection and AsCpf1 activity mediated by tandem guides wasassayed using the SURVEYOR nuclease assay.

FIG. 115B demonstrates INDEL formation in both the GRIN28 and the EMX1gene.

FIG. 116 shows FnCpf1 cleavage of an array with increasingconcentrations of EDTA (and decreasing concentrations of Mg2+). Thebuffer is 20 mM TrisHCl pH 7 (room temperature), 50 mM KCl, and includesa murine RNAse inhibitor to prevent degradation of RNA due to potentialtrace amount of non-specific RNase carried over from proteinpurification.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

The present application describes novel RNA-guided endonucleases (e.g.Cpf1 effector proteins) which are functionally distinct from theCRISPR-Cas9 systems described previously and hence the terminology ofelements associated with these novel endonulceases are modifiedaccordingly herein. Cpf1-associated CRISPR arrays described herein areprocessed into mature crRNAs without the requirement of an additionaltracrRNA. The crRNAs described herein comprise a spacer sequence (orguide sequence) and a direct repeat sequence and a Cpf1p-crRNA complexby itself is sufficient to efficiently cleave target DNA. The seedsequence described herein, e.g. the seed sequence of a FnCpf1 guide RNAis approximately within the first 5 nt on the 5′ end of the spacersequence (or guide sequence) and mutations within the seed sequenceadversely affect cleavage activity of the Cpf1 effector protein complex.

In general, a CRISPR system is characterized by elements that promotethe formation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). In the context of formation of a CRISPR complex, “targetsequence” refers to a sequence to which a guide sequence is designed totarget, e.g. have complementarity, where hybridization between a targetsequence and a guide sequence promotes the formation of a CRISPRcomplex. The section of the guide sequence through which complementarityto the target sequence is important for cleavage activity is referred toherein as the seed sequence. A target sequence may comprise anypolynucleotide, such as DNA or RNA polynucleotides and is comprisedwithin a target locus of interest. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. The hereindescribed invention encompasses novel effector proteins of Class 2CRISPR-Cas systems, of which Cas9 is an exemplary effector protein andhence terms used in this application to describe novel effectorproteins, may correlate to the terms used to describe the CRISPR-Cas9system.

The CRISPR-Cas loci has more than 50 gene families and there is nostrictly universal genes. Therefore, no single evolutionary tree isfeasible and a multi-pronged approach is needed to identify newfamilies. So far, there is comprehensive cas gene identification of 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in FIG. 1 . Class 1 includes multisubunitcrRNA-effector complexes (Cascade) and Class 2 includes Single-subunitcrRNA-effector complexes (Cas9-like). FIG. 2 provides a molecularorganization of CRISPR-Cas. FIG. 3 provides structures of Type I and IIIeffector complexes: common architecture/common ancestry despiteextensive sequence divergence. FIG. 4 shows CRISPR-Cas as a RNArecognition motif (RRM)-centered system. FIG. 5 shows Cas1 phylogenywhere recombination of adaptation and crRNA-effector modules show amajor aspect of CRISPR-Cas evolution. FIG. 6 shows a CRISPR-Cas census,specifically a distribution of CRISPR-Cas types/subtypes among archaeaand bacteria.

The action of the CRISPR-Cas system is usually divided into threestages: (1) adaptation or spacer integration, (2) processing of theprimary transcript of the CRISPR locus (pre-crRNA) and maturation of thecrRNA which includes the spacer and variable regions corresponding to 5′and 3′ fragments of CRISPR repeats, and (3) DNA (or RNA) interference.Two proteins, Cas1 and Cas2, that are present in the great majority ofthe known CRISPR-Cas systems are sufficient for the insertion of spacersinto the CRISPR cassettes. These two proteins form a complex that isrequired for this adaptation process; the endonuclease activity of Cas1is required for spacer integration whereas Cas2 appears to perform anonenzymatic function. The Cas1-Cas2 complex represents the highlyconserved “information processing” module of CRISPR-Cas that appears tobe quasi-autonomous from the rest of the system. (See Annotation andClassification of CRISPR-Cas Systems. Makarova Kans., Koonin E V.Methods Mol Biol. 2015; 1311:47-75).

The previously described Class 2 systems, namely Type II and theputative Type V, consisted of only three or four genes in the casoperon, namely the cas1 and cas2 genes comprising the adaptation module(the cas1-cas2 pair of genes are not involved in interference), a singlemultidomain effector protein that is responsible for interference butalso contributes to the pre-crRNA processing and adaptation, and often afourth gene with uncharacterized functions that is dispensable in atleast some Type II systems (and in some cases the fourth gene is cas4(biochemical or in silico evidence shows that Cas4 is a PD-(DE)xKsuperfamily nuclease with three-cysteine C-terminal cluster; possesses5′-ssDNA exonuclease activity) or csn2, which encodes an inactivatedATPase). In most cases, a CRISPR array and a gene for a distinct RNAspecies known as tracrRNA, a trans-encoded small CRISPR RNA, areadjacent to Class 2 cas operons. The tracrRNA is partially homologous tothe repeats within the respective CRISPR array and is essential for theprocessing of pre-crRNA that is catalyzed by RNAse III, a ubiquitousbacterial enzyme that is not associated with the CRISPR-Cas loci.

Cas1 is the most conserved protein that is present in most of theCRISPR-Cas systems and evolves slower than other Cas proteins.Accordingly, Cas1 phylogeny has been used as the guide for CRISPR-Cassystem classification. Biochemical or in silico evidence shows that Cas1is a metal-dependent deoxyribonuclease. Deletion of Cas1 in E. coliresults in increased sensitivity to DNA damage and impaired chromosomalsegregation as described in “A dual function of the CRISPR-Cassystem inbacterial antivirus immunity and DNA repair,” Babu M et al. MolMicrobiol 79:484-502 (2011). Biochemical or in silico evidence showsthat Cas 2 is a RNase specific to U-rich regions and is adouble-stranded DNase.

Aspects of the invention relate to the identification and engineering ofnovel effector proteins associated with Class 2 CRISPR-Cas systems. In apreferred embodiment, the effector protein comprises a single-subuniteffector module. In a further embodiment the effector protein isfunctional in prokaryotic or eukaryotic cells for in vitro, in vivo orex vivo applications. An aspect of the invention encompassescomputational methods and algorithms to predict new Class 2 CRISPR-Cassystems and identify the components therein.

In one embodiment, a computational method of identifying novel Class 2CRISPR-Cas loci comprises the following steps: detecting all contigsencoding the Cas1 protein; identifying all predicted protein codinggenes within 20 kB of the cas1 gene; comparing the identified genes withCas protein-specific profiles and predicting CRISPR arrays; selectingunclassified candidate CRISPR-Cas loci containing proteins larger than500 amino acids (>500 aa); analyzing selected candidates using PSI-BLASTand HHPred, thereby isolating and identifying novel Class 2 CRISPR-Casloci. In addition to the above mentioned steps, additional analysis ofthe candidates may be conducted by searching metagenomics databases foradditional homologs.

In one aspect the detecting all contigs encoding the Cas1 protein isperformed by GenemarkS which a gene prediction program as furtherdescribed in “GeneMarkS: a self-training method for prediction of genestarts in microbial genomes. Implications for finding sequence motifs inregulatory regions.” John Besemer, Alexandre Lomsadze and MarkBorodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, hereinincorporated by reference.

In one aspect the identifying all predicted protein coding genes iscarried out by comparing the identified genes with Cas protein-specificprofiles and annotating them according to NCBI Conserved Domain Database(CDD) which is a protein annotation resource that consists of acollection of well-annotated multiple sequence alignment models forancient domains and full-length proteins. These are available asposition-specific score matrices (PSSMs) for fast identification ofconserved domains in protein sequences via RPS-BLAST. CDD contentincludes NCBI-curated domains, which use 3D-structure information toexplicitly define domain boundaries and provide insights intosequence/structure/function relationships, as well as domain modelsimported from a number of external source databases (Pfam, SMART, COG,PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted using aPILER-CR program which is a public domain software for finding CRISPRrepeats as described in “PILER-CR: fast and accurate identification ofCRISPR repeats”, Edgar, R. C., BMC Bioinformatics, January 20; 8:18(2007), herein incorporated by reference.

In a further aspect, the case by case analysis is performed usingPSI-BLAST (Position-Specific Iterative Basic Local Alignment SearchTool). PSI-BLAST derives a position-specific scoring matrix (PSSM) orprofile from the multiple sequence alignment of sequences detected abovea given score threshold using protein-protein BLAST. This PSSM is usedto further search the database for new matches, and is updated forsubsequent iterations with these newly detected sequences. Thus,PSI-BLAST provides a means of detecting distant relationships betweenproteins.

In another aspect, the case by case analysis is performed using HHpred,a method for sequence database searching and structure prediction thatis as easy to use as BLAST or PSI-BLAST and that is at the same timemuch more sensitive in finding remote homologs. In fact, HHpred'ssensitivity is competitive with the most powerful servers for structureprediction currently available. HHpred is the first server that is basedon the pairwise comparison of profile hidden Markov models (HMMs).Whereas most conventional sequence search methods search sequencedatabases such as UniProt or the NR, HHpred searches alignmentdatabases, like Pfam or SMART. This greatly simplifies the list of hitsto a number of sequence families instead of a clutter of singlesequences. All major publicly available profile and alignment databasesare available through HHpred. HHpred accepts a single query sequence ora multiple alignment as input. Within only a few minutes it returns thesearch results in an easy-to-read format similar to that of PSI-BLAST.Search options include local or global alignment and scoring secondarystructure similarity. HHpred can produce pairwise query-templatesequence alignments, merged query-template multiple alignments (e.g. fortransitive searches), as well as 3D structural models calculated by theMODELLER software from HHpred alignments.

The term “nucleic acid-targeting system”, wherein nucleic acid is DNA orRNA, and in some aspects may also refer to DNA-RNA hybrids orderivatives thereof, refers collectively to transcripts and otherelements involved in the expression of or directing the activity of DNAor RNA-targeting CRISPR-associated (“Cas”) genes, which may includesequences encoding a DNA or RNA-targeting Cas protein and a DNA orRNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (inCRISPR-Cas9 system but not all systems) a trans-activating CRISPR-Cassystem RNA (tracrRNA) sequence, or other sequences and transcripts froma DNA or RNA-targeting CRISPR locus. In the Cpf1 DNA targetingRNA-guided endonuclease systems described herein, a tracrRNA sequence isnot required. In general, a RNA-targeting system is characterized byelements that promote the formation of a RNA-targeting complex at thesite of a target RNA sequence. In the context of formation of a DNA orRNA-targeting complex, “target sequence” refers to a DNA or RNA sequenceto which a DNA or RNA-targeting guide RNA is designed to havecomplementarity, where hybridization between a target sequence and aRNA-targeting guide RNA promotes the formation of a RNA-targetingcomplex. In some embodiments, a target sequence is located in thenucleus or cytoplasm of a cell.

In an aspect of the invention, novel DNA targeting systems also referredto as DNA-targeting CRISPR-Cas or the CRISPR-Cas DNA-targeting system ofthe present application are based on identified Type V (e.g. subtype V-Aand subtype V-B) Cas proteins which do not require the generation ofcustomized proteins to target specific DNA sequences but rather a singleeffector protein or enzyme can be programmed by a RNA molecule torecognize a specific DNA target, in other words the enzyme can berecruited to a specific DNA target using said RNA molecule. Aspects ofthe invention particularly relate to DNA targeting RNA-guided Cpf1CRISPR systems.

In an aspect of the invention, novel RNA targeting systems also referredto as RNA- or RNA-targeting CRISPR-Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific RNA target, inother words the enzyme can be recruited to a specific RNA target usingsaid RNA molecule.

The nucleic acids-targeting systems, the vector systems, the vectors andthe compositions described herein may be used in various nucleicacids-targeting applications, altering or modifying synthesis of a geneproduct, such as a protein, nucleic acids cleavage, nucleic acidsediting, nucleic acids splicing; trafficking of target nucleic acids,tracing of target nucleic acids, isolation of target nucleic acids,visualization of target nucleic acids, etc.

As used herein, a Cas protein or a CRISPR enzyme refers to any of theproteins presented in the new classification of CRISPR-Cas systems. Inan advantageous embodiment, the present invention encompasses effectorproteins identified in a Type V CRISPR-Cas loci, e.g. a Cpf1-encodingloci denoted as subtype V-A. Presently, the subtype V-A loci encompassescas1, cas2, a distinct gene denoted cpf1 and a CRISPR array. Cpf1(CRISPR-associated protein Cpf1, subtype PREFRAN) is a large protein(about 1300 amino acids) that contains a RuvC-like nuclease domainhomologous to the corresponding domain of Cas9 along with a counterpartto the characteristic arginine-rich cluster of Cas9. However, Cpf1 lacksthe HNH nuclease domain that is present in all Cas9 proteins, and theRuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9where it contains long inserts including the HNH domain. Accordingly, inparticular embodiments, the CRISPR-Cas enzyme comprises only a RuvC-likenuclease domain.

The Cpf1 gene is found in several diverse bacterial genomes, typicallyin the same locus with cas1, cas2, and cas4 genes and a CRISPR cassette(for example, FNFX1_1431-FNFX1_1428 of Francisella cf. novicida Fx1).Thus, the layout of this putative novel CRISPR-Cas system appears to besimilar to that of type II-B. Furthermore, similar to Cas9, the Cpf1protein contains a readily identifiable C-terminal region that ishomologous to the transposon ORF-B and includes an active RuvC-likenuclease, an arginine-rich region, and a Zn finger (absent in Cas9).However, unlike Cas9, Cpf1 is also present in several genomes without aCRISPR-Cas context and its relatively high similarity with ORF-Bsuggests that it might be a transposon component. It was suggested thatif this was a genuine CRISPR-Cas system and Cpf1 is a functional analogof Cas9 it would be a novel CRISPR-Cas type, namely type V (SeeAnnotation and Classification of CRISPR-Cas Systems. Makarova Kans.,Koonin E V. Methods Mol Biol. 2015; 1311:47-75). However, as describedherein, Cpf1 is denoted to be in subtype V-A to distinguish it fromC2c1p which does not have an identical domain structure and is hencedenoted to be in subtype V-B.

In an advantageous embodiment, the present invention encompassescompositions and systems comprising effector proteins identified in aCpf1 loci denoted as subtype V-A.

Aspects of the invention also encompass methods and uses of thecompositions and systems described herein in genome engineering, e.g.for altering or manipulating the expression of one or more genes or theone or more gene products, in prokaryotic or eukaryotic cells, in vitro,in vivo or ex vivo.

In embodiments of the invention the terms mature crRNA and guide RNA andsingle guide RNA are used interchangeably as in foregoing citeddocuments such as WO 2014/093622 (PCT/US2013/074667). In general, aguide sequence is any polynucleotide sequence having sufficientcomplementarity with a target polynucleotide sequence to hybridize withthe target sequence and direct sequence-specific binding of a CRISPRcomplex to the target sequence. In some embodiments, the degree ofcomplementarity between a guide sequence and its corresponding targetsequence, when optimally aligned using a suitable alignment algorithm,is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,99%, or more. Optimal alignment may be determined with the use of anysuitable algorithm for aligning sequences, non-limiting example of whichinclude the Smith-Waterman algorithm, the Needleman-Wunsch algorithm,algorithms based on the Burrows-Wheeler Transform (e.g., the BurrowsWheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (NovocraftTechnologies; available at www.novocraft.com), ELAND (Illumina, SanDiego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. Preferably the guide sequence is 10-30nucleotides long. The ability of a guide sequence to directsequence-specific binding of a CRISPR complex to a target sequence maybe assessed by any suitable assay. For example, the components of aCRISPR system sufficient to form a CRISPR complex, including the guidesequence to be tested, may be provided to a host cell having thecorresponding target sequence, such as by transfection with vectorsencoding the components of the CRISPR sequence, followed by anassessment of preferential cleavage within the target sequence, such asby Surveyor assay as described herein. Similarly, cleavage of a targetpolynucleotide sequence may be evaluated in a test tube by providing thetarget sequence, components of a CRISPR complex, including the guidesequence to be tested and a control guide sequence different from thetest guide sequence, and comparing binding or rate of cleavage at thetarget sequence between the test and control guide sequence reactions.Other assays are possible, and will occur to those skilled in the art. Aguide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a genome of acell. Exemplary target sequences include those that are unique in thetarget genome.

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Vectors for and that result inexpression in a eukaryotic cell can be referred to herein as “eukaryoticexpression vectors.” Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or“sgRNA” or “one or more nucleic acid components” of a Type V CRISPR-Caslocus effector protein comprises any polynucleotide sequence havingsufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence. In some embodiments, the degree ofcomplementarity, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X,BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).The ability of a guide sequence (within a nucleic acid-targeting guideRNA) to direct sequence-specific binding of a nucleic acid-targetingcomplex to a target nucleic acid sequence may be assessed by anysuitable assay. For example, the components of a nucleic acid-targetingCRISPR system sufficient to form a nucleic acid-targeting complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence may be evaluated in a test tube byproviding the target nucleic acid sequence, components of a nucleicacid-targeting complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art. A guide sequence, and hencea nucleic acid-targeting guide RNA may be selected to target any targetnucleic acid sequence. The target sequence may be DNA. The targetsequence may be any RNA sequence. In some embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of messenger RNA (mRNA), pre-mRNA, ribosomaal RNA (rRNA),transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double strandedRNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (lncRNA), andsmall cytoplasmatic RNA (scRNA). In some preferred embodiments, thetarget sequence may be a sequence within a RNA molecule selected fromthe group consisting of mRNA, pre-mRNA, and rRNA. In some preferredembodiments, the target sequence may be a sequence within a RNA moleculeselected from the group consisting of ncRNA, and lncRNA. In some morepreferred embodiments, the target sequence may be a sequence within anmRNA molecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide RNA is selected toreduce the degree secondary structure within the RNA-targeting guideRNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carrand GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

The “tracrRNA” sequence or analogous terms includes any polynucleotidesequence that has sufficient complementarity with a crRNA sequence tohybridize. As indicated herein above, in embodiments of the presentinvention, the tracrRNA is not required for cleavage activity of Cpf1effector protein complexes.

Applicants also perform a challenge experiment to verify the DNAtargeting and cleaving capability of a Type V/Type VI protein such asCpf1/C2c1/C2c2. This experiment closely parallels similar work in E.coli for the heterologous expression of StCas9 (Sapranauskas, R. et al.Nucleic Acids Res 39, 9275-9282 (2011)). Applicants introduce a plasmidcontaining both a PAM and a resistance gene into the heterologous E.coli, and then plate on the corresponding antibiotic. If there is DNAcleavage of the plasmid, Applicants observe no viable colonies.

In further detail, the assay is as follows for a DNA target. Two E. colistrains are used in this assay. One carries a plasmid that encodes theendogenous effector protein locus from the bacterial strain. The otherstrain carries an empty plasmid (e.g. pACYC184, control strain). Allpossible 7 or 8 bp PAM sequences are presented on an antibioticresistance plasmid (pUC19 with ampicillin resistance gene). The PAM islocated next to the sequence of proto-spacer 1 (the DNA target to thefirst spacer in the endogenous effector protein locus). Two PAMlibraries were cloned. One has a 8 random bp 5′ of the proto-spacer(e.g. total of 65536 different PAM sequences=complexity). The otherlibrary has 7 random bp 3′ of the proto-spacer (e.g. total complexity is16384 different PAMs). Both libraries were cloned to have in average 500plasmids per possible PAM. Test strain and control strain weretransformed with 5′PAM and 3′PAM library in separate transformations andtransformed cells were plated separately on ampicillin plates.Recognition and subsequent cutting/interference with the plasmid rendersa cell vulnerable to ampicillin and prevents growth. Approximately 12 hafter transformation, all colonies formed by the test and controlstrains where harvested and plasmid DNA was isolated. Plasmid DNA wasused as template for PCR amplification and subsequent deep sequencing.Representation of all PAMs in the untransfomed libraries showed theexpected representation of PAMs in transformed cells. Representation ofall PAMs found in control strains showed the actual representation.Representation of all PAMs in test strain showed which PAMs are notrecognized by the enzyme and comparison to the control strain allowsextracting the sequence of the depleted PAM.

In some embodiments of CRISPR-Cas9 systems, the degree ofcomplementarity between the tracrRNA sequence and crRNA sequence isalong the length of the shorter of the two when optimally aligned. Asdescribed herein, in embodiments of the present invention, the tracrRNAis not required. In some embodiments of previously described CRISPR-Cassystems (e.g. CRISPR-Cas9 systems), chimeric synthetic guide RNAs(sgRNAs) designs may incorporate at least 12 bp of duplex structurebetween the crRNA and tracrRNA, however in the Cpf1 CRISPR systemsdescribed herein such chimeric RNAs (chi-RNAs) are not possible as thesystem does not utilize a tracrRNA.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of nucleic acid-targeting guide RNAdelivered. Optimal concentrations of nucleic acid-targeting guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing the analyzethe extent of modification at potential off-target genomic loci. Theconcentration that gives the highest level of on-target modificationwhile minimizing the level of off-target modification should be chosenfor in vivo delivery. The nucleic acid-targeting system is derivedadvantageously from a Type V/Type VI CRISPR system. In some embodiments,one or more elements of a nucleic acid-targeting system is derived froma particular organism comprising an endogenous RNA-targeting system. Inpreferred embodiments of the invention, the RNA-targeting system is aType V/Type VI CRISPR system. In particular embodiments, the Type V/TypeVI RNA-targeting Cas enzyme is Cpf1/C2c1/C2c2. Non-limiting examples ofCas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7,Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, ormodified versions thereof. In embodiments, the Type V/Type VI proteinsuch as Cpf1/C2c1/C2c2 as referred to herein also encompasses ahomologue or an orthologue of a Type V/Type VI protein such asCpf1/C2c1/C2c2. The terms “orthologue” (also referred to as “ortholog”herein) and “homologue” (also referred to as “homolog” herein) are wellknown in the art. By means of further guidance, a “homologue” of aprotein as used herein is a protein of the same species which performsthe same or a similar function as the protein it is a homologue ofHomologous proteins may but need not be structurally related, or areonly partially structurally related. An “orthologue” of a protein asused herein is a protein of a different species which performs the sameor a similar function as the protein it is an orthologue of Orthologousproteins may but need not be structurally related, or are only partiallystructurally related. Homologs and orthologs may be identified byhomology modelling (see, e.g., Greer, Science vol. 228 (1985) 1055, andBlundell et al. Eur J Biochem vol 172 (1988), 513) or “structural BLAST”(Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a “structural BLAST”:using structural relationships to infer function. Protein Sci. 2013April; 22(4):359-66. doi: 10.1002/pro.2225). See also Shmakov et al.(2015) for application in the field of CRISPR-Cas loci. Homologousproteins may but need not be structurally related, or are only partiallystructurally related. In particular embodiments, the homologue ororthologue of Cpf1 as referred to herein has a sequence homology oridentity of at least 80%, more preferably at least 85%, even morepreferably at least 90%, such as for instance at least 95% with Cpf1. Infurther embodiments, the homologue or orthologue of Cpf1 as referred toherein has a sequence identity of at least 80%, more preferably at least85%, even more preferably at least 90%, such as for instance at least95% with the wild type Cpf1. Where the Cpf1 has one or more mutations(mutated), the homologue or orthologue of said Cpf1 as referred toherein has a sequence identity of at least 80%, more preferably at least85%, even more preferably at least 90%, such as for instance at least95% with the mutated Cpf1.

In an embodiment, the type V Cas protein may be an ortholog of anorganism of a genus which includes, but is not limited toAcidaminococcus sp, Lachnospiraceae bacterium or Moraxella bovoculi; inparticular embodiments, the type V Cas protein may be an ortholog of anorganism of a species which includes, but is not limited toAcidaminococcus sp. BV3L6; Lachnospiraceae bacterium ND2006 (LbCpf1) orMoraxella bovoculi 237. In particular embodiments, the homologue ororthologue of Cpf1 as referred to herein has a sequence homology oridentity of at least 80%, more preferably at least 85%, even morepreferably at least 90%, such as for instance at least 95% with one ormore of the Cpf1 sequences disclosed herein. In further embodiments, thehomologue or orthologue of Cpf as referred to herein has a sequenceidentity of at least 80%, more preferably at least 85%, even morepreferably at least 90%, such as for instance at least 95% with the wildtype FnCpf1, AsCpf1 or LbCpf1.

In particular embodiments, the Cpf1 protein of the invention has asequence homology or identity of at least 60%, more particularly atleast 70, such as at least 80%, more preferably at least 85%, even morepreferably at least 90%, such as for instance at least 95% with FnCpf1,AsCpf1 or LbCpf1. In further embodiments, the Cpf1 protein as referredto herein has a sequence identity of at least 60%, such as at least 70%,more particularly at least 80%, more preferably at least 85%, even morepreferably at least 90%, such as for instance at least 95% with the wildtype AsCpf1 or LbCpf1. In particular embodiments, the Cpf1 protein ofthe present invention has less than 60% sequence identity with FnCpf1.The skilled person will understand that this includes truncated forms ofthe Cpf1 protein whereby the sequence identity is determined over thelength of the truncated form.

Some methods of identifying orthologs of CRISPR-Cas system enzymes mayinvolve identifying tracr sequences in genomes of interest.Identification of tracr sequences may relate to the following steps:Search for the direct repeats or tracr mate sequences in a database toidentify a CRISPR region comprising a CRISPR enzyme. Search forhomologous sequences in the CRISPR region flanking the CRISPR enzyme inboth the sense and antisense directions. Look for transcriptionalterminators and secondary structures. Identify any sequence that is nota direct repeat or a tracr mate sequence but has more than 50% identityto the direct repeat or tracr mate sequence as a potential tracrsequence. Take the potential tracr sequence and analyze fortranscriptional terminator sequences associated therewith. In thissystem, RNA-sequencing data revealed that the potential tracrRNAsidentified computationally were only lowly expressed suggestingpossibility that tracrRNA may not be necessary for function of thepresent system. After further evaluation of the FnCpf1 locus andaddition of in vitro cleavage results, Applicants concluded that targetDNA cleavage by a Cpf1 effector protein complex does not require atracrRNA. Applicants determined that Cpf1 effector protein complexescomprising only a Cpf1 effector protein and a crRNA (guide RNAcomprising a direct repeat sequence and a guide sequence) weresufficient to cleave target DNA.

It will be appreciated that any of the functionalities described hereinmay be engineered into CRISPR enzymes from other orthologs, includingchimeric enzymes comprising fragments from multiple orthologs. Examplesof such orthologs are described elsewhere herein. Thus, chimeric enzymesmay comprise fragments of CRISPR enzyme orthologs of organisms of agenus which includes but is not limited to Corynebacter, Sutterella,Legionella, Treponema, Filifactor, Eubacterium, Streptococcus,Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium,Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia,Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma andCampylobacter. A chimeric enzyme can comprise a first fragment and asecond fragment, and the fragments can be of CRISPR enzyme orthologs oforganisms of genuses herein mentioned or of species herein mentioned;advantageously the fragments are from CRISPR enzyme orthologs ofdifferent species.

In embodiments, the Type V/Type VI RNA-targeting effector protein, inparticular the Cpf1/C2c1/C2c2 protein as referred to herein alsoencompasses a functional variant of Cpf1/C2c1/C2c2 or a homologue or anorthologue thereof. A “functional variant” of a protein as used hereinrefers to a variant of such protein which retains at least partially theactivity of that protein. Functional variants may include mutants (whichmay be insertion, deletion, or replacement mutants), includingpolymorphs, etc. Also included within functional variants are fusionproducts of such protein with another, usually unrelated, nucleic acid,protein, polypeptide or peptide. Functional variants may be naturallyoccurring or may be man-made. Advantageous embodiments can involveengineered or non-naturally occurring Type V/Type VI RNA-targetingeffector protein, e.g., Cpf1/C2c1/C2c2 or an ortholog or homologthereof.

In an embodiment, nucleic acid molecule(s) encoding the Type V/Type VIRNA-targeting effector protein, in particular Cpf1/C2c1/C2c2 or anortholog or homolog thereof, may be codon-optimized for expression in aneukaryotic cell. A eukaryote can be as herein discussed. Nucleic acidmolecule(s) can be engineered or non-naturally occurring.

In an embodiment, the Type V/Type VI RNA-targeting effector protein, inparticular Cpf1/C2c1/C2c2 or an ortholog or homolog thereof, maycomprise one or more mutations (and hence nucleic acid molecule(s)coding for same may have mutation(s)). The mutations may be artificiallyintroduced mutations and may include but are not limited to one or moremutations in a catalytic domain. Examples of catalytic domains withreference to a Cas9 enzyme may include but are not limited to RuvC I,RuvC II, RuvC III and HNH domains.

In an embodiment, the Type V/Type VI protein such as Cpf1/C2c1/C2c2 oran ortholog or homolog thereof, may be used as a generic nucleic acidbinding protein with fusion to or being operably linked to a functionaldomain. Exemplary functional domains may include but are not limited totranslational initiator, translational activator, translationalrepressor, nucleases, in particular ribonucleases, a spliceosome, beads,a light inducible/controllable domain or a chemicallyinducible/controllable domain.

In some embodiments, the unmodified nucleic acid-targeting effectorprotein may have cleavage activity. In some embodiments, theRNA-targeting effector protein may direct cleavage of one or bothnucleic acid (DNA or RNA) strands at the location of or near a targetsequence, such as within the target sequence and/or within thecomplement of the target sequence or at sequences associated with thetarget sequence. In some embodiments, the nucleic acid-targetingeffector protein may direct cleavage of one or both DNA or RNA strandswithin about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200,500, or more base pairs from the first or last nucleotide of a targetsequence. In some embodiments, the cleavage may be staggered, i.e.generating sticky ends. In some embodiments, the cleavage is a staggeredcut with a 5′ overhang. In some embodiments, the cleavage is a staggeredcut with a 5′ overhang of 1 to 5 nucleotides, preferably of 4 or 5nucleotides. In some embodiments, the cleavage site is distant from thePAM, e.g., the cleavage occurs after the 18^(th) nucleotide on thenon-target strand and after the 23^(rd) nucleotide on the targetedstrand (FIG. 97A). In some embodiments, the cleavage site occurs afterthe 18^(th) nucleotide (counted from the PAM) on the non-target strandand after the 23^(rd) nucleotide (counted from the PAM) on the targetedstrand (FIG. 97A). In some embodiments, a vector encodes a nucleicacid-targeting effector protein that may be mutated with respect to acorresponding wild-type enzyme such that the mutated nucleicacid-targeting effector protein lacks the ability to cleave one or bothDNA or RNA strands of a target polynucleotide containing a targetsequence. As a further example, two or more catalytic domains of a Casprotein (e.g. RuvC I, RuvC II, and RuvC III or the HNH domain of a Cas9protein) may be mutated to produce a mutated Cas protein substantiallylacking all DNA cleavage activity. As described herein, correspondingcatalytic domains of a Cpf1 effector protein may also be mutated toproduce a mutated Cpf1 effector protein lacking all DNA cleavageactivity or having substantially reduced DNA cleavage activity. In someembodiments, a nucleic acid-targeting effector protein may be consideredto substantially lack all RNA cleavage activity when the RNA cleavageactivity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%,0.1%, 0.01%, or less of the nucleic acid cleavage activity of thenon-mutated form of the enzyme; an example can be when the nucleic acidcleavage activity of the mutated form is nil or negligible as comparedwith the non-mutated form. An effector protein may be identified withreference to the general class of enzymes that share homology to thebiggest nuclease with multiple nuclease domains from the Type V/Type VICRISPR system. Most preferably, the effector protein is a Type V/Type VIprotein such as Cpf1/C2c1/C2c2. In further embodiments, the effectorprotein is a Type V protein. By derived, Applicants mean that thederived enzyme is largely based, in the sense of having a high degree ofsequence homology with, a wildtype enzyme, but that it has been mutated(modified) in some way as known in the art or as described herein.

Again, it will be appreciated that the terms Cas and CRISPR enzyme andCRISPR protein and Cas protein are generally used interchangeably and atall points of reference herein refer by analogy to novel CRISPR effectorproteins further described in this application, unless otherwiseapparent, such as by specific reference to Cas9. As mentioned above,many of the residue numberings used herein refer to the effector proteinfrom the Type V/Type VI CRISPR locus. However, it will be appreciatedthat this invention includes many more effector proteins from otherspecies of microbes. In certain embodiments, effector proteins may beconstitutively present or inducibly present or conditionally present oradministered or delivered. Effector protein optimization may be used toenhance function or to develop new functions, one can generate chimericeffector proteins. And as described herein effector proteins may bemodified to be used as a generic nucleic acid binding proteins.

Typically, in the context of a nucleic acid-targeting system, formationof a nucleic acid-targeting complex (comprising a guide RNA hybridizedto a target sequence and complexed with one or more nucleicacid-targeting effector proteins) results in cleavage of one or both DNAor RNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, or more base pairs from) the target sequence. As used herein theterm “sequence(s) associated with a target locus of interest” refers tosequences near the vicinity of the target sequence (e.g. within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the targetsequence, wherein the target sequence is comprised within a target locusof interest).

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667) as an example of a codonoptimized sequence (from knowledge in the art and this disclosure, codonoptimizing coding nucleic acid molecule(s), especially as to effectorprotein (e.g., Cpf1) is within the ambit of the skilled artisan). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a DNA/RNA-targeting Casprotein is codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a plant or a mammal, including but notlimited to human, or non-human eukaryote or animal or mammal as hereindiscussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammalor primate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g., about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at www.kazusa.orjp/codon/and these tables canbe adapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, P A), arealso available. In some embodiments, one or more codons (e.g., 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga DNA/RNA-targeting Cas protein corresponds to the most frequently usedcodon for a particular amino acid. As to codon usage in yeast, referenceis made to the online Yeast Genome database available athttp://www.yeastgenome.org/community/codon_usage.shtml, or Codonselection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar. 25;257(6):3026-31. As to codon usage in plants including algae, referenceis made to Codon usage in higher plants, green algae, and cyanobacteria,Campbell and Gowri, Plant Physiol. 1990 January; 92(1): 1-11; as well asCodon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan.25; 17(2):477-98; or Selection on the codon bias of chloroplast andcyanelle genes in different plant and algal lineages, Morton B R, J MolEvol. 1998 April; 46(4):449-59.

In some embodiments, a vector encodes a nucleic acid-targeting effectorprotein such as the Type V/Type VI RNA-targeting effector protein, inparticular Cpf1/C2c1/C2c2 or an ortholog or homolog thereof comprisingone or more nuclear localization sequences (NLSs), such as about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In someembodiments, the RNA-targeting effector protein comprises about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near theamino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more NLSs at or near the carboxy-terminus, or a combination of these(e.g., zero or at least one or more NLS at the amino-terminus and zeroor at one or more NLS at the carboxy terminus). When more than one NLSis present, each may be selected independently of the others, such thata single NLS may be present in more than one copy and/or in combinationwith one or more other NLSs present in one or more copies. In someembodiments, an NLS is considered near the N- or C-terminus when thenearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 40, 50, or more amino acids along the polypeptide chain from theN- or C-terminus. Non-limiting examples of NLSs include an NLS sequencederived from: the NLS of the SV40 virus large T-antigen, having theamino acid sequence PKKKRKV (SEQ ID NO: 2); the NLS from nucleoplasmin(e.g., the nucleoplasmin bipartite NLS with the sequenceKRPAATKKAGQAKKKK (SEQ ID NO: 3)); the c-myc NLS having the amino acidsequence PAAKRVKLD (SEQ ID NO: 4) or RQRRNELKRSP (SEQ ID NO: 5); thehRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 6); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV(SEQ ID NO: 7) of the IBB domain from importin-alpha; the sequencesVSRKRPRP (SEQ ID NO: 8) and PPKKARED (SEQ ID NO: 9) of the myoma Tprotein; the sequence PQPKKKPL (SEQ ID NO: 10) of human p53; thesequence SALIKKKKKMAP (SEQ ID NO: 11) of mouse c-abl IV; the sequencesDRLRR (SEQ ID NO: 12) and PKQKKRK (SEQ ID NO: 13) of the influenza virusNS1; the sequence RKLKKKIKKL (SEQ ID NO: 14) of the Hepatitis virusdelta antigen; the sequence REKKKFLKRR (SEQ ID NO: 15) of the mouse Mx1protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 16) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 17) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, the one or more NLSs are of sufficient strength to driveaccumulation of the DNA/RNA-targeting Cas protein in a detectable amountin the nucleus of a eukaryotic cell. In general, strength of nuclearlocalization activity may derive from the number of NLSs in the nucleicacid-targeting effector protein, the particular NLS(s) used, or acombination of these factors. Detection of accumulation in the nucleusmay be performed by any suitable technique. For example, a detectablemarker may be fused to the nucleic acid-targeting protein, such thatlocation within a cell may be visualized, such as in combination with ameans for detecting the location of the nucleus (e.g., a stain specificfor the nucleus such as DAPI). Cell nuclei may also be isolated fromcells, the contents of which may then be analyzed by any suitableprocess for detecting protein, such as immunohistochemistry, Westernblot, or enzyme activity assay. Accumulation in the nucleus may also bedetermined indirectly, such as by an assay for the effect of nucleicacid-targeting complex formation (e.g., assay for DNA or RNA cleavage ormutation at the target sequence, or assay for altered gene expressionactivity affected by DNA or RNA-targeting complex formation and/or DNAor RNA-targeting Cas protein activity), as compared to a control notexposed to the nucleic acid-targeting Cas protein or nucleicacid-targeting complex, or exposed to a nucleic acid-targeting Casprotein lacking the one or more NLSs. In preferred embodiments of theherein described Cpf1 effector protein complexes and systems the codonoptimized Cpf1 effector proteins comprise an NLS attached to theC-terminal of the protein. In certain embodiments, other localizationtags may be fused to the Cas protein, such as without limitation forlocalizing the Cas to particular sites in a cell, such as organells,such mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear orcellular) membranes, ribosomes, nucleoluse, ER, cytoskeleton, vacuoles,centrosome, nucleosome, granules, centrioles, etc

In some embodiments, one or more vectors driving expression of one ormore elements of a nucleic acid-targeting system are introduced into ahost cell such that expression of the elements of the nucleicacid-targeting system direct formation of a nucleic acid-targetingcomplex at one or more target sites. For example, a nucleicacid-targeting effector enzyme and a nucleic acid-targeting guide RNAcould each be operably linked to separate regulatory elements onseparate vectors. RNA(s) of the nucleic acid-targeting system can bedelivered to a transgenic nucleic acid-targeting effector protein animalor mammal, e.g., an animal or mammal that constitutively or inducibly orconditionally expresses nucleic acid-targeting effector protein; or ananimal or mammal that is otherwise expressing nucleic acid-targetingeffector proteins or has cells containing nucleic acid-targetingeffector proteins, such as by way of prior administration thereto of avector or vectors that code for and express in vivo nucleicacid-targeting effector proteins. Alternatively, two or more of theelements expressed from the same or different regulatory elements, maybe combined in a single vector, with one or more additional vectorsproviding any components of the nucleic acid-targeting system notincluded in the first vector. nucleic acid-targeting system elementsthat are combined in a single vector may be arranged in any suitableorientation, such as one element located 5′ with respect to (“upstream”of) or 3′ with respect to (“downstream” of) a second element. The codingsequence of one element may be located on the same or opposite strand ofthe coding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a nucleic acid-targeting effectorprotein and the nucleic acid-targeting guide RNA, embedded within one ormore intron sequences (e.g., each in a different intron, two or more inat least one intron, or all in a single intron). In some embodiments,the nucleic acid-targeting effector protein and the nucleicacid-targeting guide RNA may be operably linked to and expressed fromthe same promoter. Delivery vehicles, vectors, particles, nanoparticles,formulations and components thereof for expression of one or moreelements of a nucleic acid-targeting system are as used in the foregoingdocuments, such as WO 2014/093622 (PCT/US2013/074667). In someembodiments, a vector comprises one or more insertion sites, such as arestriction endonuclease recognition sequence (also referred to as a“cloning site”). In some embodiments, one or more insertion sites (e.g.,about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreinsertion sites) are located upstream and/or downstream of one or moresequence elements of one or more vectors. When multiple different guidesequences are used, a single expression construct may be used to targetnucleic acid-targeting activity to multiple different, correspondingtarget sequences within a cell. For example, a single vector maycomprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,or more guide sequences. In some embodiments, about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containingvectors may be provided, and optionally delivered to a cell. In someembodiments, a vector comprises a regulatory element operably linked toan enzyme-coding sequence encoding a nucleic acid-targeting effectorprotein. Nucleic acid-targeting effector protein or nucleicacid-targeting guide RNA or RNA(s) can be delivered separately; andadvantageously at least one of these is delivered via a particlecomplex. nucleic acid-targeting effector protein mRNA can be deliveredprior to the nucleic acid-targeting guide RNA to give time for nucleicacid-targeting effector protein to be expressed. Nucleic acid-targetingeffector protein mRNA might be administered 1-12 hours (preferablyaround 2-6 hours) prior to the administration of nucleic acid-targetingguide RNA. Alternatively, nucleic acid-targeting effector protein mRNAand nucleic acid-targeting guide RNA can be administered together.Advantageously, a second booster dose of guide RNA can be administered1-12 hours (preferably around 2-6 hours) after the initialadministration of nucleic acid-targeting effector protein mRNA+guideRNA. Additional administrations of nucleic acid-targeting effectorprotein mRNA and/or guide RNA might be useful to achieve the mostefficient levels of genome modification.

In one aspect, the invention provides methods for using one or moreelements of a nucleic acid-targeting system. The nucleic acid-targetingcomplex of the invention provides an effective means for modifying atarget DNA or RNA (single or double stranded, linear or super-coiled).The nucleic acid-targeting complex of the invention has a wide varietyof utility including modifying (e.g., deleting, inserting,translocating, inactivating, activating) a target DNA or RNA in amultiplicity of cell types. As such the nucleic acid-targeting complexof the invention has a broad spectrum of applications in, e.g., genetherapy, drug screening, disease diagnosis, and prognosis. An exemplarynucleic acid-targeting complex comprises a DNA or RNA-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin the target locus of interest.

In one embodiment, this invention provides a method of cleaving a targetRNA. The method may comprise modifying a target RNA using a nucleicacid-targeting complex that binds to the target RNA and effect cleavageof said target RNA. In an embodiment, the nucleic acid-targeting complexof the invention, when introduced into a cell, may create a break (e.g.,a single or a double strand break) in the RNA sequence. For example, themethod can be used to cleave a disease RNA in a cell. For example, anexogenous RNA template comprising a sequence to be integrated flanked byan upstream sequence and a downstream sequence may be introduced into acell. The upstream and downstream sequences share sequence similaritywith either side of the site of integration in the RNA. Where desired, adonor RNA can be mRNA. The exogenous RNA template comprises a sequenceto be integrated (e.g., a mutated RNA). The sequence for integration maybe a sequence endogenous or exogenous to the cell. Examples of asequence to be integrated include RNA encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction. The upstream and downstream sequences in the exogenous RNAtemplate are selected to promote recombination between the RNA sequenceof interest and the donor RNA. The upstream sequence is a RNA sequencethat shares sequence similarity with the RNA sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is aRNA sequence that shares sequence similarity with the RNA sequencedownstream of the targeted site of integration. The upstream anddownstream sequences in the exogenous RNA template can have 75%, 80%,85%, 90%, 95%, or 100% sequence identity with the targeted RNA sequence.Preferably, the upstream and downstream sequences in the exogenous RNAtemplate have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identitywith the targeted RNA sequence. In some methods, the upstream anddownstream sequences in the exogenous RNA template have about 99% or100% sequence identity with the targeted RNA sequence. An upstream ordownstream sequence may comprise from about 20 bp to about 2500 bp, forexample, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, or 2500 bp. In some methods, the exemplary upstream ordownstream sequence have about 200 bp to about 2000 bp, about 600 bp toabout 1000 bp, or more particularly about 700 bp to about 1000 bp. Insome methods, the exogenous RNA template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous RNA template of theinvention can be constructed using recombinant techniques (see, forexample, Sambrook et al., 2001 and Ausubel et al., 1996). In a methodfor modifying a target RNA by integrating an exogenous RNA template, abreak (e.g., double or single stranded break in double or singlestranded DNA or RNA) is introduced into the DNA or RNA sequence by thenucleic acid-targeting complex, the break is repaired via homologousrecombination with an exogenous RNA template such that the template isintegrated into the RNA target. The presence of a double-stranded breakfacilitates integration of the template. In other embodiments, thisinvention provides a method of modifying expression of a RNA in aeukaryotic cell. The method comprises increasing or decreasingexpression of a target polynucleotide by using a nucleic acid-targetingcomplex that binds to the DNA or RNA (e.g., mRNA or pre-mRNA). In somemethods, a target RNA can be inactivated to effect the modification ofthe expression in a cell. For example, upon the binding of aRNA-targeting complex to a target sequence in a cell, the target RNA isinactivated such that the sequence is not translated, the coded proteinis not produced, or the sequence does not function as the wild-typesequence does. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. The target RNA of a RNA-targeting complex can be anyRNA endogenous or exogenous to the eukaryotic cell. For example, thetarget RNA can be a RNA residing in the nucleus of the eukaryotic cell.The target RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA). Examples of target RNA include a sequence associatedwith a signaling biochemical pathway, e.g., a signaling biochemicalpathway-associated RNA. Examples of target RNA include a diseaseassociated RNA. A “disease-associated” RNA refers to any RNA which isyielding translation products at an abnormal level or in an abnormalform in cells derived from a disease-affected tissues compared withtissues or cells of a non disease control. It may be a RNA transcribedfrom a gene that becomes expressed at an abnormally high level; it maybe a RNA transcribed from a gene that becomes expressed at an abnormallylow level, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated RNA also refersto a RNA transcribed from a gene possessing mutation(s) or geneticvariation that is directly responsible or is in linkage disequilibriumwith a gene(s) that is responsible for the etiology of a disease. Thetranslated products may be known or unknown, and may be at a normal orabnormal level. The target RNA of a RNA-targeting complex can be any RNAendogenous or exogenous to the eukaryotic cell. For example, the targetRNA can be a RNA residing in the nucleus of the eukaryotic cell. Thetarget RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA).

In some embodiments, the method may comprise allowing a nucleicacid-targeting complex to bind to the target DNA or RNA to effectcleavage of said target DNA or RNA thereby modifying the target DNA orRNA, wherein the nucleic acid-targeting complex comprises a nucleicacid-targeting effector protein complexed with a guide RNA hybridized toa target sequence within said target DNA or RNA. In one aspect, theinvention provides a method of modifying expression of DNA or RNA in aeukaryotic cell. In some embodiments, the method comprises allowing anucleic acid-targeting complex to bind to the DNA or RNA such that saidbinding results in increased or decreased expression of said DNA or RNA;wherein the nucleic acid-targeting complex comprises a nucleicacid-targeting effector protein complexed with a guide RNA. Similarconsiderations and conditions apply as above for methods of modifying atarget DNA or RNA. In fact, these sampling, culturing andre-introduction options apply across the aspects of the presentinvention. In one aspect, the invention provides for methods ofmodifying a target DNA or RNA in a eukaryotic cell, which may be invivo, ex vivo or in vitro. In some embodiments, the method comprisessampling a cell or population of cells from a human or non-human animal,and modifying the cell or cells. Culturing may occur at any stage exvivo. The cell or cells may even be re-introduced into the non-humananimal or plant. For re-introduced cells it is particularly preferredthat the cells are stem cells.

Indeed, in any aspect of the invention, the nucleic acid-targetingcomplex may comprise a nucleic acid-targeting effector protein complexedwith a guide RNA hybridized to a target sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving DNA or RNA sequence targeting, that relate to the nucleicacid-targeting system and components thereof. In advantageousembodiments, the effector enzyme is a Type V/Type VI protein such asCpf1/C2c1/C2c2. An advantage of the present methods is that the CRISPRsystem minimizes or avoids off-target binding and its resulting sideeffects. This is achieved using systems arranged to have a high degreeof sequence specificity for the target DNA or RNA.

In relation to a nucleic acid-targeting complex or system preferably,the crRNA sequence has one or more stem loops or hairpins and is 30 ormore nucleotides in length, 40 or more nucleotides in length, or 50 ormore nucleotides in length; the crRNA sequence is between 10 to 30nucleotides in length, the nucleic acid-targeting effector protein is aType V/Type VI Cas enzyme. In certain embodiments, the crRNA sequence isbetween 42 and 44 nucleotides in length, and the nucleic acid-targetingCas protein is Cpf1 of Francisella tularensis subsp.novocida U112. Incertain embodiments, the crRNA comprises, consists essentially of, orconsists of 19 nucleotides of a direct repeat and between 23 and 25nucleotides of spacer sequence, and the nucleic acid-targeting Casprotein is Cpf1 of Francisella tularensis subsp.novocida U112.

The use of two different aptamers (each associated with a distinctnucleic acid-targeting guide RNAs) allows an activator-adaptor proteinfusion and a repressor-adaptor protein fusion to be used, with differentnucleic acid-targeting guide RNAs, to activate expression of one DNA orRNA, whilst repressing another. They, along with their different guideRNAs can be administered together, or substantially together, in amultiplexed approach. A large number of such modified nucleicacid-targeting guide RNAs can be used all at the same time, for example10 or 20 or 30 and so forth, whilst only one (or at least a minimalnumber) of effector protein molecules need to be delivered, as acomparatively small number of effector protein molecules can be usedwith a large number modified guides. The adaptor protein may beassociated (preferably linked or fused to) one or more activators or oneor more repressors. For example, the adaptor protein may be associatedwith a first activator and a second activator. The first and secondactivators may be the same, but they are preferably differentactivators. Three or more or even four or more activators (orrepressors) may be used, but package size may limit the number beinghigher than 5 different functional domains. Linkers are preferably used,over a direct fusion to the adaptor protein, where two or morefunctional domains are associated with the adaptor protein. Suitablelinkers might include the GlySer linker.

It is also envisaged that the nucleic acid-targeting effectorprotein-guide RNA complex as a whole may be associated with two or morefunctional domains. For example, there may be two or more functionaldomains associated with the nucleic acid-targeting effector protein, orthere may be two or more functional domains associated with the guideRNA (via one or more adaptor proteins), or there may be one or morefunctional domains associated with the nucleic acid-targeting effectorprotein and one or more functional domains associated with the guide RNA(via one or more adaptor proteins).

The fusion between the adaptor protein and the activator or repressormay include a linker. For example, GlySer linkers GGGS (SEQ ID NO: 18)can be used. They can be used in repeats of 3 ((GGGGS)₃ (SEQ ID NO: 19))or 6 (SEQ ID NO: 20), 9 (SEQ ID NO: 21) or even 12 (SEQ ID NO: 22) ormore, to provide suitable lengths, as required. Linkers can be usedbetween the guide RNAs and the functional domain (activator orrepressor), or between the nucleic acid-targeting Cas protein (Cas) andthe functional domain (activator or repressor). The linkers the user toengineer appropriate amounts of “mechanical flexibility”.

The invention comprehends a nucleic acid-targeting complex comprising anucleic acid-targeting effector protein and a guide RNA, wherein thenucleic acid-targeting effector protein comprises at least one mutation,such that the nucleic acid-targeting effector protein has no more than5% of the activity of the nucleic acid-targeting effector protein nothaving the at least one mutation and, optional, at least one or morenuclear localization sequences; the guide RNA comprises a guide sequencecapable of hybridizing to a target sequence in a RNA of interest in acell; and wherein: the nucleic acid-targeting effector protein isassociated with two or more functional domains; or at least one loop ofthe guide RNA is modified by the insertion of distinct RNA sequence(s)that bind to one or more adaptor proteins, and wherein the adaptorprotein is associated with two or more functional domains; or thenucleic acid-targeting Cas protein is associated with one or morefunctional domains and at least one loop of the guide RNA is modified bythe insertion of distinct RNA sequence(s) that bind to one or moreadaptor proteins, and wherein the adaptor protein is associated with oneor more functional domains.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated an increase in the risk of havingor developing a disease. In some embodiments, the method comprises (a)introducing one or more vectors into a eukaryotic cell, wherein the oneor more vectors drive expression of one or more of: a Cpf1 enzyme and aprotected guide RNA comprising a guide sequence linked to a directrepeat sequence; and (b) allowing a CRISPR complex to bind to a targetpolynucleotide to effect cleavage of the target polynucleotide withinsaid disease gene, wherein the CRISPR complex comprises the Cpf1 enzymecomplexed with the guide RNA comprising the sequence that is hybridizedto the target sequence within the target polynucleotide, therebygenerating a model eukaryotic cell comprising a mutated disease gene. Insome embodiments, said cleavage comprises cleaving one or two strands atthe location of the target sequence by said Cpf1 enzyme. In someembodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by non-homologous end joining(NHEJ)-based gene insertion mechanisms with an exogenous templatepolynucleotide, wherein said repair results in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of saidtarget polynucleotide. In some embodiments, said mutation results in oneor more amino acid changes in a protein expression from a genecomprising the target sequence.

In an aspect the invention provides methods as herein discussed whereinthe host is a eukaryotic cell. In an aspect the invention provides amethod as herein discussed wherein the host is a mammalian cell. In anaspect the invention provides a method as herein discussed, wherein thehost is a non-human eukaryote cell. In an aspect the invention providesa method as herein discussed, wherein the non-human eukaryote cell is anon-human mammal cell. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal cell may be including,but not limited to, primate bovine, ovine, procine, canine, rodent,Leporidae such as monkey, cow, sheep, pig, dog, rabbit, rat or mousecell. In an aspect the invention provides a method as herein discussed,the cell may be a non-mammalian eukaryotic cell such as poultry bird(e.g., chicken), vertebrate fish (e.g., salmon) or shellfish (e.g.,oyster, claim, lobster, shrimp) cell. In an aspect the inventionprovides a method as herein discussed, the non-human eukaryote cell is aplant cell. The plant cell may be of a monocot or dicot or of a crop orgrain plant such as cassava, corn, sorghum, soybean, wheat, oat or rice.The plant cell may also be of an algae, tree or production plant, fruitor vegetable (e.g., trees such as citrus trees, e.g., orange, grapefruitor lemon trees; peach or nectarine trees; apple or pear trees; nut treessuch as almond or walnut or pistachio trees; nightshade plants; plantsof the genus Brassica; plants of the genus Lactuca; plants of the genusSpinacia; plants of the genus Capsicum; cotton, tobacco, asparagus,carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper,lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape,coffee, cocoa, etc).

In one aspect, the invention provides a method for developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. In some embodiments, a disease gene isany gene associated an increase in the risk of having or developing adisease. In some embodiments, the method comprises (a) contacting a testcompound with a model cell of any one of the above-describedembodiments; and (b) detecting a change in a readout that is indicativeof a reduction or an augmentation of a cell signaling event associatedwith said mutation in said disease gene, thereby developing saidbiologically active agent that modulates said cell signaling eventassociated with said disease gene.

In one aspect the invention provides for a method of selecting one ormore cell(s) by introducing one or more mutations in a gene in the oneor more cell (s), the method comprising: introducing one or more vectorsinto the cell (s), wherein the one or more vectors drive expression ofone or more of: Cpf1, a guide sequence linked to a direct repeatsequence, and an editing template; wherein the editing templatecomprises the one or more mutations that abolish Cpf1 cleavage; allowinghomologous recombination of the editing template with the targetpolynucleotide in the cell(s) to be selected; allowing a Cpf1 CRISPR-Cascomplex to bind to a target polynucleotide to effect cleavage of thetarget polynucleotide within said gene, wherein the Cpf1 CRISPR-Cascomplex comprises the Cpf1 complexed with (1) the guide sequence that ishybridized to the target sequence within the target polynucleotide, and(2) the direct repeat sequence, wherein binding of the Cpf1 CRISPR-Cascomplex to the target polynucleotide induces cell death, therebyallowing one or more cell(s) in which one or more mutations have beenintroduced to be selected; this includes the present split Cpf1. Inanother preferred embodiment of the invention the cell to be selectedmay be a eukaryotic cell. Aspects of the invention allow for selectionof specific cells without requiring a selection marker or a two-stepprocess that may include a counter-selection system. In particularembodiments, the model eukaryotic cell is comprised within a modeleukaryotic organism.

In one aspect, the invention provides a recombinant polynucleotidecomprising a guide sequence downstream of a direct repeat sequence,wherein the guide sequence when expressed directs sequence-specificbinding of a Cpf1 CRISPR-Cas complex to a corresponding target sequencepresent in a eukaryotic cell. In some embodiments, the target sequenceis a viral sequence present in a eukaryotic cell. In some embodiments,the target sequence is a proto-oncogene or an oncogene.

In one aspect, the invention provides a vector system or eukaryotic hostcell comprising (a) a first regulatory element operably linked to adirect repeat sequence and one or more insertion sites for inserting oneor more guide sequences (including any of the modified guide sequencesas described herein) downstream of the DR sequence, wherein whenexpressed, the guide sequence directs sequence-specific binding of aCpf1 CRISPR-Cas complex to a target sequence in a eukaryotic cell,wherein the Cpf1 CRISPR-Cas complex comprises Cpf1 (including any of themodified enzymes as described herein) complexed with the guide sequencethat is hybridized to the target sequence (and optionally the DRsequence); and/or (b) a second regulatory element operably linked to anenzyme-coding sequence encoding said Cpf1 enzyme comprising a nuclearlocalization sequence and/or NES. In some embodiments, the host cellcomprises components (a) and (b). In some embodiments, component (a),component (b), or components (a) and (b) are stably integrated into agenome of the host eukaryotic cell. In some embodiments, component (a)further comprises two or more guide sequences operably linked to thefirst regulatory element, wherein when expressed, each of the two ormore guide sequences direct sequence specific binding of a Cpf1CRISPR-Cas complex to a different target sequence in a eukaryotic cell.In some embodiments, the CRISPR enzyme comprises one or more nuclearlocalization sequences and/or nuclear export sequences or NES ofsufficient strength to drive accumulation of said CRISPR enzyme in adetectable amount in and/or out of the nucleus of a eukaryotic cell. Insome embodiments, the Cpf1 enzyme is derived from Francisella tularensis1, Francisella tularensis sub sp. novicida, Prevotella albensis,Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus,Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacteriumGW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6,Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum,Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai,Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3,Prevotella disiens, or Porphyromonas macacae Cpf1, including any of themodified enzymes as described herein, and may include further alterationor mutation of the Cpf1, and can be a chimeric Cpf1. In someembodiments, the CRISPR enzyme is codon-optimized for expression in aeukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavageof one or two strands at the location of the target sequence. In apreferred embodiment, the strand break is a staggered cut with a 5′overhang. In some embodiments, the Cpf1 lacks DNA strand cleavageactivity (e.g., no more than 5% nuclease activity as compared with awild type enzyme or enzyme not having the mutation or alteration thatdecreases nuclease activity). In some embodiments, the first regulatoryelement is a polymerase III promoter. In some embodiments, the secondregulatory element is a polymerase II promoter. In some embodiments, thedirect repeat has a minimum length of 16 nts and a single stem loop. Infurther embodiments the direct repeat has a length longer than 16 nts,preferably more than 17 nts, and has more than one stem loop oroptimized secondary structures. In some embodiments, the guide sequenceis at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, orbetween 16-25, or between 16-20 nucleotides in length.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system or host cell as described herein and instructions forusing the kit.

Modified Cpf1 Enzymes

Computational analysis of the primary structure of Cpf1 nucleasesreveals three distinct regions (FIG. 1 ). First a C-terminal RuvC likedomain, which is the only functional characterized domain. Second aN-terminal alpha-helical region and thirst a mixed alpha and betaregion, located between the RuvC like domain and the alpha-helicalregion.

Several small stretches of unstructured regions are predicted within theCpf1 primary structure. Unstructured regions, which are exposed to thesolvent and not conserved within different Cpf1 orthologs, are preferredsides for splits and insertions of small protein sequences (FIGS. 2 and3 ). In addition, these sides can be used to generate chimeric proteinsbetween Cpf1 orthologs.

Based on the above information, mutants can be generated which lead toinactivation of the enzyme or which modify the double strand nuclease tonickase activity. In alternative embodiments, this information is usedto develop enzymes with reduced off-target effects (described elsewhereherein)

In certain of the above-described Cpf1 enzymes, the enzyme is modifiedby mutation of one or more residues including but not limited topositions D917, E1006, E1028, D1227, D1255A, N1257, according to FnCpf1protein or any corresponding ortholog. In an aspect the inventionprovides a herein-discussed composition wherein the Cpf1 enzyme is aninactivated enzyme which comprises one or more mutations selected fromthe group consisting of D917A, E1006A, E1028A, D1227A, D1255A, N1257A,D917A, E1006A, E1028A, D1227A, D1255A and N1257A according to FnCpf1protein or corresponding positions in a Cpf1 ortholog. In an aspect theinvention provides a herein-discussed composition, wherein the CRISPRenzyme comprises D917, or E1006 and D917, or D917 and D1255, accordingto FnCpf1 protein or a corresponding position in a Cpf1 ortholog.

In certain of the above-described Cpf1 enzymes, the enzyme is modifiedby mutation of one or more residues (in the RuvC domain) including butnot limited to positions R909, R912, R930, R947, K949, R951, R955, K965,K968, K1000, K1002, R1003, K1009, K1017, K1022, K1029, K1035, K1054,K1072, K1086, R1094, K1095, K1109, K1118, K1142, K1150, K1158, K1159,R1220, R1226, R1242, and/or R1252 with reference to amino acid positionnumbering of AsCpf1 (Acidaminococcus sp. BV3L6).

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of one or more residues (inthe RAD50) domain including but not limited positions K324, K335, K337,R331, K369, K370, R386, R392, R393, K400, K404, K406, K408, K414, K429,K436, K438, K459, K460, K464, R670, K675, R681, K686, K689, R699, K705,R725, K729, K739, K748, and/or K752 with reference to amino acidposition numbering of AsCpf1 (Acidaminococcus sp. BV3L6).

In certain of the Cpf1 enzymes, the enzyme is modified by mutation ofone or more residues including but not limited positions R912, T923,R947, K949, R951, R955, K965, K968, K1000, R1003, K1009, K1017, K1022,K1029, K1072, K1086, F1103, R1226, and/or R1252 with reference to aminoacid position numbering of AsCpf1 (Acidaminococcus sp. BV3L6).

In certain embodiments, the Cpf1 enzyme is modified by mutation of oneor more residues including but not limited positions R833, R836, K847,K879, K881, R883, R887, K897, K900, K932, R935, K940, K948, K953, K960,K984, K1003, K1017, R1033, R1138, R1165, and/or R1252 with reference toamino acid position numbering of LbCpf1 (Lachnospiraceae bacteriumND2006).

In certain embodiments, the Cpf1 enzyme is modified by mutation of oneor more residues including but not limited positions K15, R18, K26, Q34,R43, K48, K51, R56, R84, K85, K87, N93, R103, N104, T118, K123, K134,R176, K177, R192, K200, K226, K273, K275, T291, R301, K307, K369, S404,V409, K414, K436, K438, K468, D482, K516, R518, K524, K530, K532, K548,K559, K570, R574, K592, D596, K603, K607, K613, C647, R681, K686, H720,K739, K748, K757, T766, K780, R790, P791, K796, K809, K815, T816, K860,R862, R863, K868, K897, R909, R912, T923, R947, K949, R951, R955, K965,K968, K1000, R1003, K1009, K1017, K1022, K1029, A1053, K1072, K1086,F1103, S1209, R1226, R1252, K1273, K1282, and/or K1288 with reference toamino acid position numbering of AsCpf1 (Acidaminococcus sp. BV3L6).

In certain embodiments, the enzyme is modified by mutation of one ormore residues including but not limited positions K15, R18, K26, R34,R43, K48, K51, K56, K87, K88, D90, K96, K106, K107, K120, Q125, K143,R186, K187, R202, K210, K235, K296, K298, K314, K320, K326, K397, K444,K449, E454, A483, E491, K527, K541, K581, R583, K589, K595, K597, K613,K624, K635, K639, K656, K660, K667, K671, K677, K719, K725, K730, K763,K782, K791, R800, K809, K823, R833, K834, K839, K852, K858, K859, K869,K871, R872, K877, K905, R918, R921, K932, 1960, K962, R964, R968, K978,K981, K1013, R1016, K1021, K1029, K1034, K1041, K1065, K1084, and/orK1098 with reference to amino acid position numbering of FnCpf1(Francisella novicida U112).

In certain embodiments, the enzyme is modified by mutation of one ormore residues including but not limited positions K15, R18, K26, K34,R43, K48, K51, R56, K83, K84, R86, K92, R102, K103, K116, K121, R158,E159, R174, R182, K206, K251, K253, K269, K271, K278, P342, K380, R385,K390, K415, K421, K457, K471, A506, R508, K514, K520, K522, K538, Y548,K560, K564, K580, K584, K591, K595, K601, K634, K640, R645, K679, K689,K707, T716, K725, R737, R747, R748, K753, K768, K774, K775, K785, K787,R788, Q793, K821, R833, R836, K847, K879, K881, R883, R887, K897, K900,K932, R935, K940, K948, K953, K960, K984, K1003, K1017, R1033, K1121,R1138, R1165, K1190, K1199, and/or K1208 with reference to amino acidposition numbering of LbCpf1 (Lachnospiraceae bacterium ND2006).

In certain embodiments, the enzyme is modified by mutation of one ormore residues including but not limited positions K14, R17, R25, K33,M42, Q47, K50, D55, K85, N86, K88, K94, R104, K105, K118, K123, K131,R174, K175, R190, R198, I221, K267, Q269, K285, K291, K297, K357, K403,K409, K414, K448, K460, K501, K515, K550, R552, K558, K564, K566, K582,K593, K604, K608, K623, K627, K633, K637, E643, K780, Y787, K792, K830,Q846, K858, K867, K876, K890, R900, K901, M906, K921, K927, K928, K937,K939, R940, K945, Q975, R987, R990, K1001, R1034, 11036, R1038, R1042,K1052, K1055, K1087, R1090, K1095, N1103, K1108, K1115, K1139, K1158,R1172, K1188, K1276, R1293, A1319, K1340, K1349, and/or K1356 withreference to amino acid position numbering of MbCpf1 (Moraxella bovoculi237).

Deactivated/Inactivated Cpf1 Protein

Where the Cpf1 protein has nuclease activity, the Cpf1 protein may bemodified to have diminished nuclease activity e.g., nucleaseinactivation of at least 70%, at least 80%, at least 90%, at least 95%,at least 97%, or 100% as compared with the wild type enzyme; or to putin another way, a Cpf1 enzyme having advantageously about 0% of thenuclease activity of the non-mutated or wild type Cpf1 enzyme or CRISPRenzyme, or no more than about 3% or about 5% or about 10% of thenuclease activity of the non-mutated or wild type Cpf1 enzyme, e.g. ofthe non-mutated or wild type Francisella novicida U112 (FnCpf1),Acidaminococcus sp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006(LbCpf1) or Moraxella bovoculi 237 (MbCpf1 Cpf1 enzyme or CRISPR enzyme.This is possible by introducing mutations into the nuclease domains ofthe Cpf1 and orthologs thereof.

More particularly, the inactivated Cpf1 enzymes include enzymes mutatedin amino acid positions As908, As993, As1263 of AsCpf1 or correspondingpositions in Cpf1 orthologs. Additionally, the inactivated Cpf1 enzymesinclude enzymes mutated in amino acid position Lb832, 925, 947 or 1180of LbCpf1 or corresponding positions in Cpf1 orthologs. Moreparticularly, the inactivated Cpf1 enzymes include enzymes comprisingone or more of mutations AsD908A, AsE993A, AsD1263A of AsCpf1 orcorresponding mutations in Cpf1 orthologs. Additionally, the inactivatedCpf1 enzymes include enzymes comprising one or more of mutationsLbD832A, E925A, D947A or D1180A of LbCpf1 or corresponding mutations inCpf1 orthologs.

The inactivated Cpf1 CRISPR enzyme may have associated (e.g., via fusionprotein) one or more functional domains, including for example, one ormore domains from the group comprising, consisting essentially of, orconsisting of methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity, DNA cleavage activity, nucleic acid binding activity, andmolecular switches (e.g., light inducible). Preferred domains are Fok1,VP64, P65, HSF1, MyoD1. In the event that Fok1 is provided, it isadvantageous that multiple Fok1 functional domains are provided to allowfor a functional dimer and that gRNAs are designed to provide properspacing for functional use (Fok1) as specifically described in Tsai etal. Nature Biotechnology, Vol. 32, Number 6, June 2014). The adaptorprotein may utilize known linkers to attach such functional domains. Insome cases it is advantageous that additionally at least one NLS isprovided. In some instances, it is advantageous to position the NLS atthe N terminus. When more than one functional domain is included, thefunctional domains may be the same or different.

In general, the positioning of the one or more functional domain on theinactivated Cpf1 enzyme is one which allows for correct spatialorientation for the functional domain to affect the target with theattributed functional effect. For example, if the functional domain is atranscription activator (e.g., VP64 or p65), the transcription activatoris placed in a spatial orientation which allows it to affect thetranscription of the target. Likewise, a transcription repressor will beadvantageously positioned to affect the transcription of the target, anda nuclease (e.g., Fok1) will be advantageously positioned to cleave orpartially cleave the target. This may include positions other than theN-/C-terminus of the CRISPR enzyme.

Destabilized Cpf1

In certain embodiments, the effector protein (CRISPR enzyme; Cpf1)according to the invention as described herein is associated with orfused to a destabilization domain (DD). In some embodiments, the DD isER50. A corresponding stabilizing ligand for this DD is, in someembodiments, 4HT. As such, in some embodiments, one of the at least oneDDs is ER50 and a stabilizing ligand therefor is 4HT or CMP8. In someembodiments, the DD is DHFR50. A corresponding stabilizing ligand forthis DD is, in some embodiments, TMP. As such, in some embodiments, oneof the at least one DDs is DHFR50 and a stabilizing ligand therefor isTMP. In some embodiments, the DD is ER50. A corresponding stabilizingligand for this DD is, in some embodiments, CMP8. CMP8 may therefore bean alternative stabilizing ligand to 4HT in the ER50 system. While itmay be possible that CMP8 and 4HT can/should be used in a competitivematter, some cell types may be more susceptible to one or the other ofthese two ligands, and from this disclosure and the knowledge in the artthe skilled person can use CMP8 and/or 4HT.

In some embodiments, one or two DDs may be fused to the N-terminal endof the CRISPR enzyme with one or two DDs fused to the C-terminal of theCRISPR enzyme. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are the same DD, i.e. the DDs arehomologous. Thus, both (or two or more) of the DDs could be ER50 DDs.This is preferred in some embodiments. Alternatively, both (or two ormore) of the DDs could be DHFR50 DDs. This is also preferred in someembodiments. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are different DDs, i.e. the DDs areheterologous. Thus, one of the DDS could be ER50 while one or more ofthe DDs or any other DDs could be DHFR50. Having two or more DDs whichare heterologous may be advantageous as it would provide a greater levelof degradation control. A tandem fusion of more than one DD at the N orC-term may enhance degradation; and such a tandem fusion can be, forexample ER50-ER50-C2c2 or DHFR-DHFR-Cpf1. It is envisaged that highlevels of degradation would occur in the absence of either stabilizingligand, intermediate levels of degradation would occur in the absence ofone stabilizing ligand and the presence of the other (or another)stabilizing ligand, while low levels of degradation would occur in thepresence of both (or two of more) of the stabilizing ligands. Controlmay also be imparted by having an N-terminal ER50 DD and a C-terminalDHFR50 DD.

In some embodiments, the fusion of the CRISPR enzyme with the DDcomprises a linker between the DD and the CRISPR enzyme. In someembodiments, the linker is a GlySer linker. In some embodiments, theDD-CRISPR enzyme further comprises at least one Nuclear Export Signal(NES). In some embodiments, the DD-CRISPR enzyme comprises two or moreNESs. In some embodiments, the DD-CRISPR enzyme comprises at least oneNuclear Localization Signal (NLS). This may be in addition to an NES. Insome embodiments, the CRISPR enzyme comprises or consists essentially ofor consists of a localization (nuclear import or export) signal as, oras part of, the linker between the CRISPR enzyme and the DD. HA or Flagtags are also within the ambit of the invention as linkers. Applicantsuse NLS and/or NES as linker and also use Glycine Serine linkers asshort as GS up to (GGGGS)3.

Destabilizing domains have general utility to confer instability to awide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar. 7,2012; 134(9): 3942-3945, incorporated herein by reference. CMP8 or4-hydroxytamoxifen can be destabilizing domains. More generally, Atemperature-sensitive mutant of mammalian DHFR (DHFRts), a destabilizingresidue by the N-end rule, was found to be stable at a permissivetemperature but unstable at 37° C. The addition of methotrexate, ahigh-affinity ligand for mammalian DHFR, to cells expressing DHFRtsinhibited degradation of the protein partially. This was an importantdemonstration that a small molecule ligand can stabilize a proteinotherwise targeted for degradation in cells. A rapamycin derivative wasused to stabilize an unstable mutant of the FRB domain of mTOR (FRB*)and restore the function of the fused kinase, GSK-3β.6,7 This systemdemonstrated that ligand-dependent stability represented an attractivestrategy to regulate the function of a specific protein in a complexbiological environment. A system to control protein activity can involvethe DD becoming functional when the ubiquitin complementation occurs byrapamycin induced dimerization of FK506-binding protein and FKBP12.Mutants of human FKBP12 or ecDHFR protein can be engineered to bemetabolically unstable in the absence of their high-affinity ligands,Shield-1 or trimethoprim (TMP), respectively. These mutants are some ofthe possible destabilizing domains (DDs) useful in the practice of theinvention and instability of a DD as a fusion with a CRISPR enzymeconfers to the CRISPR protein degradation of the entire fusion proteinby the proteasome. Shield-1 and TMP bind to and stabilize the DD in adose-dependent manner. The estrogen receptor ligand binding domain(ERLBD, residues 305-549 of ERS1) can also be engineered as adestabilizing domain. Since the estrogen receptor signaling pathway isinvolved in a variety of diseases such as breast cancer, the pathway hasbeen widely studied and numerous agonist and antagonists of estrogenreceptor have been developed. Thus, compatible pairs of ERLBD and drugsare known. There are ligands that bind to mutant but not wild-type formsof the ERLBD. By using one of these mutant domains encoding threemutations (L384M, M421G, G521R)12, it is possible to regulate thestability of an ERLBD-derived DD using a ligand that does not perturbendogenous estrogen-sensitive networks. An additional mutation (Y5375)can be introduced to further destabilize the ERLBD and to configure itas a potential DD candidate. This tetra-mutant is an advantageous DDdevelopment. The mutant ERLBD can be fused to a CRISPR enzyme and itsstability can be regulated or perturbed using a ligand, whereby theCRISPR enzyme has a DD. Another DD can be a 12-kDa (107-amino-acid) tagbased on a mutated FKBP protein, stabilized by Shield1 ligand; see,e.g., Nature Methods 5, (2008). For instance a DD can be a modifiedFK506 binding protein 12 (FKBP12) that binds to and is reversiblystabilized by a synthetic, biologically inert small molecule, Shield-1;see, e.g., Banaszynski L A, Chen L C, Maynard-Smith L A, Ooi A G,Wandless T J. A rapid, reversible, and tunable method to regulateprotein function in living cells using synthetic small molecules. Cell.2006; 126:995-1004; Banaszynski L A, Sellmyer M A, Contag C H, WandlessT J, Thorne S H. Chemical control of protein stability and function inliving mice. Nat Med. 2008; 14:1123-1127; Maynard-Smith L A, Chen L C,Banaszynski L A, Ooi A G, Wandless T J. A directed approach forengineering conditional protein stability using biologically silentsmall molecules. The Journal of biological chemistry. 2007;282:24866-24872; and Rodriguez, Chem Biol. Mar. 23, 2012; 19(3):391-398—all of which are incorporated herein by reference and may beemployed in the practice of the invention in selected a DD to associatewith a CRISPR enzyme in the practice of this invention. As can be seen,the knowledge in the art includes a number of DDs, and the DD can beassociated with, e.g., fused to, advantageously with a linker, to aCRISPR enzyme, whereby the DD can be stabilized in the presence of aligand and when there is the absence thereof the DD can becomedestabilized, whereby the CRISPR enzyme is entirely destabilized, or theDD can be stabilized in the absence of a ligand and when the ligand ispresent the DD can become destabilized; the DD allows the CRISPR enzymeand hence the CRISPR-Cas complex or system to be regulated orcontrolled—turned on or off so to speak, to thereby provide means forregulation or control of the system, e.g., in an in vivo or in vitroenvironment. For instance, when a protein of interest is expressed as afusion with the DD tag, it is destabilized and rapidly degraded in thecell, e.g., by proteasomes. Thus, absence of stabilizing ligand leads toa D associated Cas being degraded. When a new DD is fused to a proteinof interest, its instability is conferred to the protein of interest,resulting in the rapid degradation of the entire fusion protein. Peakactivity for Cas is sometimes beneficial to reduce off-target effects.Thus, short bursts of high activity are preferred. The present inventionis able to provide such peaks. In some senses the system is inducible.In some other senses, the system repressed in the absence of stabilizingligand and de-repressed in the presence of stabilizing ligand.

Enzyme Mutations Reducing Off-Target Effects

In one aspect, the invention provides a non-naturally occurring orengineered CRISPR enzyme, preferably a class 2 CRISPR enzyme, preferablya Type V or VI CRISPR enzyme as described herein, such as preferably,but without limitation Cpf1 as described herein elsewhere, having one ormore mutations resulting in reduced off-target effects, i.e. improvedCRISPR enzymes for use in effecting modifications to target loci butwhich reduce or eliminate activity towards off-targets, such as whencomplexed to guide RNAs, as well as improved improved CRISPR enzymes forincreasing the activity of CRISPR enzymes, such as when complexed withguide RNAs. It is to be understood that mutated enzymes as describedherein below may be used in any of the methods according to theinvention as described herein elsewhere. Any of the methods, products,compositions and uses as described herein elsewhere are equallyapplicable with the mutated CRISPR enzymes as further detailed below. Itis to be understood, that in the aspects and embodiments as describedherein, when referring to or reading on Cpf1 as the CRISPR enzyme,reconstitution of a functional CRISPR-Cas system preferably does notrequire or is not dependent on a tracr sequence and/or direct repeat is5′ (upstream) of the guide (target or spacer) sequence.

By means of further guidance, the following particular aspects andembodiments are provided.

The inventors have surprisingly determined that modifications may bemade to CRISPR enzymes which confer reduced off-target activity comparedto unmodified CRISPR enzymes and/or increased target activity comparedto unmodified CRISPR enzymes. Thus, in certain aspects of the inventionprovided herein are improved CRISPR enzymes which may have utility in awide range of gene modifying applications. Also provided herein areCRISPR complexes, compositions and systems, as well as methods and uses,all comprising the herein disclosed modified CRISPR enzymes.

In this disclosure, the term “Cas” can mean “Cpf1” or a CRISPR enzyme.In the context of this aspect of the invention, a Cpf1 or CRISPR enzymeis mutated or modified, “whereby the enzyme in the CRISPR complex hasreduced capability of modifying one or more off-target loci as comparedto an unmodified enzyme” (or like expressions); and, when reading thisspecification, the terms “Cpf1” or “Cas” or “CRISPR enzyme and the likeare meant to include mutated or modified Cpf1 or Cas or CRISPR enzyme inaccordance with the invention, i.e., “whereby the enzyme in the CRISPRcomplex has reduced capability of modifying one or more off-target locias compared to an unmodified enzyme” (or like expressions).

In an aspect, there is provided an engineered Cpf1 protein as definedherein, such as Cpf1, wherein the protein complexes with a nucleic acidmolecule comprising RNA to form a CRISPR complex, wherein when in theCRISPR complex, the nucleic acid molecule targets one or more targetpolynucleotide loci, the protein comprises at least one modificationcompared to unmodified Cpf1 protein, and wherein the CRISPR complexcomprising the modified protein has altered activity as compared to thecomplex comprising the unmodified Cpf1 protein. It is to be understoodthat when referring herein to CRISPR “protein”, the Cpf1 proteinpreferably is a modified CRISPR enzyme (e.g. having increased ordecreased (or no) enzymatic activity, such as without limitationincluding Cpf1. The term “CRISPR protein” may be used interchangeablywith “CRISPR enzyme”, irrespective of whether the CRISPR protein hasaltered, such as increased or decreased (or no) enzymatic activity,compared to the wild type CRISPR protein.

In an aspect, the altered activity of the engineered CRISPR proteincomprises an altered binding property as to the nucleic acid moleculecomprising RNA or the target polynucleotide loci, altered bindingkinetics as to the nucleic acid molecule comprising RNA or the targetpolynucleotide loci, or altered binding specificity as to the nucleicacid molecule comprising RNA or the target polynucleotide loci comparedto off-target polynucleotide loci.

In some embodiments, the unmodified Cas has DNA cleavage activity, suchas Cpf1. In some embodiments, the Cas directs cleavage of one or bothstrands at the location of a target sequence, such as within the targetsequence and/or within the complement of the target sequence. In someembodiments, the Cas directs cleavage of one or both strands withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, ormore base pairs from the first or last nucleotide of a target sequence.In some embodiments, a vector encodes a Cas that is mutated to withrespect to a corresponding wild-type enzyme such that the mutated Caslacks the ability to cleave one or both strands of a targetpolynucleotide containing a target sequence. In some embodiments, a Casis considered to substantially lack all DNA cleavage activity when theDNA cleavage activity of the mutated enzyme is about no more than 25%,10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of thenon-mutated form of the enzyme; an example can be when the DNA cleavageactivity of the mutated form is nil or negligible as compared with thenon-mutated form. Thus, the Cas may comprise one or more mutations andmay be used as a generic DNA binding protein with or without fusion to afunctional domain. The mutations may be artificially introducedmutations or gain- or loss-of-function mutations. In one aspect of theinvention, the Cas enzyme may be fused to a protein, e.g., a TAG, and/oran inducible/controllable domain such as a chemicallyinducible/controllable domain. The Cas in the invention may be achimeric Cas proteins; e.g., a Cas having enhanced function by being achimera. Chimeric Cas proteins may be new Cas containing fragments frommore than one naturally occurring Cas. These may comprise fusions ofN-terminal fragment(s) of one Cas9 homolog with C-terminal fragment(s)of another Cas homolog. The Cas can be delivered into the cell in theform of mRNA. The expression of Cas can be under the control of aninducible promoter. It is explicitly an object of the invention to avoidreading on known mutations. Indeed, the phrase “whereby the enzyme inthe CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme and/or whereby theenzyme in the CRISPR complex has increased capability of modifying theone or more target loci as compared to an unmodified enzyme” (or likeexpressions) is not intended to read upon mutations that only result ina nickase or dead Cas or known Cas9 mutations. HOWEVER, this is not tosay that the instant invention modification(s) or mutation(s) “wherebythe enzyme in the CRISPR complex has reduced capability of modifying oneor more off-target loci as compared to an unmodified enzyme and/orwhereby the enzyme in the CRISPR complex has increased capability ofmodifying the one or more target loci as compared to an unmodifiedenzyme” (or like expressions) cannot be combined with mutations thatresult in the enzyme being a nickase or dead. Such a dead enzyme can bean enhanced nucleic acid molecule binder. And such a nickase can be anenhanced nickase. For instance, changing neutral amino acid(s) in and/ornear the groove and/or other charged residues in other locations in Casthat are in close proximity to a nucleic acid (e.g., DNA, cDNA, RNA,gRNA to positive charged amino acid(s) may result in “whereby the enzymein the CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme and/or whereby theenzyme in the CRISPR complex has increased capability of modifying theone or more target loci as compared to an unmodified enzyme”, e.g., morecutting. As this can be both enhanced on- and off-target cutting (asuper cutting Cpf1), using such with what is known in the art as atru-guide or tru-sgRNAs (see, e.g., Fu et al., “Improving CRISPR-Casnuclease specificity using truncated guide RNAs,” Nature Biotechnology32, 279-284 (2014) doi:10.1038/nbt.2808 Received 17 Nov. 2013 Accepted 6Jan. 2014 Published online 26 Jan. 2014 Corrected online 29 Jan. 2014)to have enhanced on target activity without higher off target cutting orfor making super cutting nickases, or for combination with a mutationthat renders the Cas dead for a super binder.

In certain embodiments, the altered activity of the engineered Cpf1protein comprises increased targeting efficiency or decreased off-targetbinding. In certain embodiments, the altered activity of the engineeredCpf1 protein comprises modified cleavage activity.

In certain embodiments, the altered activity comprises altered bindingproperty as to the nucleic acid molecule comprising RNA or the targetpolynucleotide loci, altered binding kinetics as to the nucleic acidmolecule comprising RNA or the target polynucleotide loci, or alteredbinding specificity as to the nucleic acid molecule comprising RNA orthe target polynucleotide loci compared to off-target polynucleotideloci.

In certain embodiments, the altered activity comprises increasedtargeting efficiency or decreased off-target binding. In certainembodiments, the altered activity comprises modified cleavage activity.In certain embodiments, the altered activity comprises increasedcleavage activity as to the target polynucleotide loci. In certainembodiments, the altered activity comprises decreased cleavage activityas to the target polynucleotide loci. In certain embodiments, thealtered activity comprises decreased cleavage activity as to off-targetpolynucleotide loci. In certain embodiments, the altered activitycomprises increased cleavage activity as to off-target polynucleotideloci.

Accordingly, in certain embodiments, there is increased specificity fortarget polynucleotide loci as compared to off-target polynucleotideloci. In other embodiments, there is reduced specificity for targetpolynucleotide loci as compared to off-target polynucleotide loci.

In an aspect of the invention, the altered activity of the engineeredCpf1 protein comprises altered helicase kinetics.

In an aspect of the invention, the engineered Cpf1 protein comprises amodification that alters association of the protein with the nucleicacid molecule comprising RNA, or a strand of the target polynucleotideloci, or a strand of off-target polynucleotide loci. In an aspect of theinvention, the engineered Cpf1 protein comprises a modification thatalters formation of the CRISPR complex.

In certain embodiments, the modified Cpf1 protein comprises amodification that alters targeting of the nucleic acid molecule to thepolynucleotide loci. In certain embodiments, the modification comprisesa mutation in a region of the protein that associates with the nucleicacid molecule. In certain embodiments, the modification comprises amutation in a region of the protein that associates with a strand of thetarget polynucleotide loci. In certain embodiments, the modificationcomprises a mutation in a region of the protein that associates with astrand of the off-target polynucleotide loci. In certain embodiments,the modification or mutation comprises decreased positive charge in aregion of the protein that associates with the nucleic acid moleculecomprising RNA, or a strand of the target polynucleotide loci, or astrand of off-target polynucleotide loci. In certain embodiments, themodification or mutation comprises decreased negative charge in a regionof the protein that associates with the nucleic acid molecule comprisingRNA, or a strand of the target polynucleotide loci, or a strand ofoff-target polynucleotide loci. In certain embodiments, the modificationor mutation comprises increased positive charge in a region of theprotein that associates with the nucleic acid molecule comprising RNA,or a strand of the target polynucleotide loci, or a strand of off-targetpolynucleotide loci. In certain embodiments, the modification ormutation comprises increased negative charge in a region of the proteinthat associates with the nucleic acid molecule comprising RNA, or astrand of the target polynucleotide loci, or a strand of off-targetpolynucleotide loci. In certain embodiments, the modification ormutation increases steric hindrance between the protein and the nucleicacid molecule comprising RNA, or a strand of the target polynucleotideloci, or a strand of off-target polynucleotide loci. In certainembodiments, the modification or mutation comprises a substitution ofLys, His, Arg, Glu, Asp, Ser, Gly, or Thr. In certain embodiments, themodification or mutation comprises a substitution with Gly, Ala, Ile,Glu, or Asp. In certain embodiments, the modification or mutationcomprises an amino acid substitution in a binding groove.

In as aspect, the present invention provides:

-   -   a non-naturally-occurring CRISPR enzyme as defined herein, such        as Cpf1,    -   wherein:

the enzyme complexes with guide RNA to form a CRISPR complex,

when in the CRISPR complex, the guide RNA targets one or more targetpolynucleotide loci and the enzyme alters the polynucleotide loci, and

the enzyme comprises at least one modification,

-   -   whereby the enzyme in the CRISPR complex has reduced capability        of modifying one or more off-target loci as compared to an        unmodified enzyme, and/or whereby the enzyme in the CRISPR        complex has increased capability of modifying the one or more        target loci as compared to an unmodified enzyme.

In any such non-naturally-occurring CRISPR enzyme, the modification maycomprise modification of one or more amino acid residues of the enzyme.

In any such non-naturally-occurring CRISPR enzyme, the modification maycomprise modification of one or more amino acid residues located in aregion which comprises residues which are positively charged in theunmodified enzyme.

In any such non-naturally-occurring CRISPR enzyme, the modification maycomprise modification of one or more amino acid residues which arepositively charged in the unmodified enzyme.

In any such non-naturally-occurring CRISPR enzyme, the modification maycomprise modification of one or more amino acid residues which are notpositively charged in the unmodified enzyme.

The modification may comprise modification of one or more amino acidresidues which are uncharged in the unmodified enzyme.

The modification may comprise modification of one or more amino acidresidues which are negatively charged in the unmodified enzyme.

The modification may comprise modification of one or more amino acidresidues which are hydrophobic in the unmodified enzyme.

The modification may comprise modification of one or more amino acidresidues which are polar in the unmodified enzyme.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the modification may comprise modification of one or moreresidues located in a groove.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the modification may comprise modification of one or moreresidues located outside of a groove.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the modification comprises a modification of one or moreresidues wherein the one or more residues comprises arginine, histidineor lysine.

In any of the above-described non-naturally-occurring CRISPR enzymes,the enzyme may be modified by mutation of said one or more residues.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an alanine residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with aspartic acid or glutamic acid.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with serine, threonine, asparagine orglutamine.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with alanine, glycine, isoleucine, leucine,methionine, phenylalanine, tryptophan, tyrosine or valine.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with a polar amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an amino acid residue which is not a polaramino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with a negatively charged amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an amino acid residue which is not anegatively charged amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an uncharged amino acid residue

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an amino acid residue which is not anuncharged amino acid residue.

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with a hydrophobic amino acid residue

In certain of the above-described non-naturally-occurring CRISPRenzymes, the enzyme is modified by mutation of said one or moreresidues, and wherein the mutation comprises substitution of a residuein the unmodified enzyme with an amino acid residue which is not ahydrophobic amino acid residue.

In some embodiments, the CRISPR enzyme, such as preferably Cpf1 enzymeis derived Francisella tularensis 1, Francisella tularensis subsp.novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1,Butyrivibrio proteoclasticus, Peregrinibacteria bacteriumGW2011_GWA2_33_10, Parcubacteria bacterium GW2011 GWC2_44_17, Smithellasp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020,Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxellabovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006,Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonasmacacae Cpf1 (e.g., a Cpf1 of one of these organisms modified asdescribed herein), and may include further mutations or alterations orbe a chimeric Cpf1.

In certain embodiments, the Cpf1 protein comprises one or more nuclearlocalization signal (NLS) domains. In certain embodiments, the Cpf1protein comprises at least two or more NLSs.

In certain embodiments, the Cpf1 protein comprises a chimeric CRISPRprotein, comprising a first fragment from a first CRISPR orthologue anda second fragment from a second CIRSPR orthologue, and the first andsecond CRISPR orthologues are different.

In certain embodiments, the enzyme is modified by or comprisesmodification, e.g., comprises, consists essentially of or consists ofmodification by mutation of any one of the residues listed herein or acorresponding residue in the respective orthologue; or the enzymecomprises, consists essentially of or consists of modification in anyone (single), two (double), three (triple), four (quadruple) or moreposition(s) in accordance with the disclosure throughout thisapplication, or a corresponding residue or position in the CRISPR enzymeorthologue, e.g., an enzyme comprising, consisting essentially of orconsisting of modification in any one of the Cpf1 residues recitedherein, or a corresponding residue or position in the CRISPR enzymeorthologue. In such an enzyme, each residue may be modified bysubstitution with an alanine residue.

Applicants recently described a method for the generation of Cas9orthologues with enhanced specificity (Slaymaker et al. 2015 “Rationallyengineered Cas9 nucleases with improved specificity”). This strategy canbe used to enhance the specificity of Cpf1 orthologues. Primary residuesfor mutagenesis are preferably all positive charges residues within theRuvC domain. Additional residues are positive charged residues that areconserved between different orthologues.

In certain embodiments, specificity of Cpf1 may be improved by mutatingresidues that stabilize the non-targeted DNA strand.

In certain of the above-described non-naturally-occurring Cpf1 enzymes,the enzyme is modified by mutation of one or more residues (in the RuvCdomain) including but not limited positions R909, R912, R930, R947,K949, R951, R955, K965, K968, K1000, K1002, R1003, K1009, K1017, K1022,K1029, K1035, K1054, K1072, K1086, R1094, K1095, K1109, K1118, K1142,K1150, K1158, K1159, R1220, R1226, R1242, and/or R1252 with reference toamino acid position numbering of AsCpf1 (Acidaminococcus sp. BV3L6).

In certain of the above-described non-naturally-occurring Cpf1 enzymes,the enzyme is modified by mutation of one or more residues (in theRAD50) domain including but not limited positions K324, K335, K337,R331, K369, K370, R386, R392, R393, K400, K404, K406, K408, K414, K429,K436, K438, K459, K460, K464, R670, K675, R681, K686, K689, R699, K705,R725, K729, K739, K748, and/or K752 with reference to amino acidposition numbering of AsCpf1 (Acidaminococcus sp. BV3L6).

In certain of the above-described non-naturally-occurring Cpf1 enzymes,the enzyme is modified by mutation of one or more residues including butnot limited positions R912, T923, R947, K949, R951, R955, K965, K968,K1000, R1003, K1009, K1017, K1022, K1029, K1072, K1086, F1103, R1226,and/or R1252 with reference to amino acid position numbering of AsCpf1(Acidaminococcus sp. BV3L6).

In certain embodiments, the enzyme is modified by mutation of one ormore residues including but not limited positions R833, R836, K847,K879, K881, R883, R887, K897, K900, K932, R935, K940, K948, K953, K960,K984, K1003, K1017, R1033, R1138, R1165, and/or R1252 with reference toamino acid position numbering of LbCpf1 (Lachnospiraceae bacteriumND2006).

In certain embodiments, the Cpf1 enzyme is modified by mutation of oneor more residues including but not limited positions K15, R18, K26, Q34,R43, K48, K51, R56, R84, K85, K87, N93, R103, N104, T118, K123, K134,R176, K177, R192, K200, K226, K273, K275, T291, R301, K307, K369, S404,V409, K414, K436, K438, K468, D482, K516, R518, K524, K530, K532, K548,K559, K570, R574, K592, D596, K603, K607, K613, C647, R681, K686, H720,K739, K748, K757, T766, K780, R790, P791, K796, K809, K815, T816, K860,R862, R863, K868, K897, R909, R912, T923, R947, K949, R951, R955, K965,K968, K1000, R1003, K1009, K1017, K1022, K1029, A1053, K1072, K1086,F1103, S1209, R1226, R1252, K1273, K1282, and/or K1288 with reference toamino acid position numbering of AsCpf1 (Acidaminococcus sp. BV3L6).

In certain embodiments, the Cpf1 enzyme is modified by mutation of oneor more residues including but not limited positions K15, R18, K26, R34,R43, K48, K51, K56, K87, K88, D90, K96, K106, K107, K120, Q125, K143,R186, K187, R202, K210, K235, K296, K298, K314, K320, K326, K397, K444,K449, E454, A483, E491, K527, K541, K581, R583, K589, K595, K597, K613,K624, K635, K639, K656, K660, K667, K671, K677, K719, K725, K730, K763,K782, K791, R800, K809, K823, R833, K834, K839, K852, K858, K859, K869,K871, R872, K877, K905, R918, R921, K932, I960, K962, R964, R968, K978,K981, K1013, R1016, K1021, K1029, K1034, K1041, K1065, K1084, and/orK1098 with reference to amino acid position numbering of FnCpf1(Francisella novicida U112).

In certain embodiments, the Cpf1 enzyme is modified by mutation of oneor more residues including but not limited positions K15, R18, K26, K34,R43, K48, K51, R56, K83, K84, R86, K92, R102, K103, K116, K121, R158,E159, R174, R182, K206, K251, K253, K269, K271, K278, P342, K380, R385,K390, K415, K421, K457, K471, A506, R508, K514, K520, K522, K538, Y548,K560, K564, K580, K584, K591, K595, K601, K634, K640, R645, K679, K689,K707, T716, K725, R737, R747, R748, K753, K768, K774, K775, K785, K787,R788, Q793, K821, R833, R836, K847, K879, K881, R883, R887, K897, K900,K932, R935, K940, K948, K953, K960, K984, K1003, K1017, R1033, K1121,R1138, R1165, K1190, K1199, and/or K1208 with reference to amino acidposition numbering of LbCpf1 (Lachnospiraceae bacterium ND2006).

In certain embodiments, the enzyme is modified by mutation of one ormore residues including but not limited positions K14, R17, R25, K33,M42, Q47, K50, D55, K85, N86, K88, K94, R104, K105, K118, K123, K131,R174, K175, R190, R198, 1221, K267, Q269, K285, K291, K297, K357, K403,K409, K414, K448, K460, K501, K515, K550, R552, K558, K564, K566, K582,K593, K604, K608, K623, K627, K633, K637, E643, K780, Y787, K792, K830,Q846, K858, K867, K876, K890, R900, K901, M906, K921, K927, K928, K937,K939, R940, K945, Q975, R987, R990, K1001, R1034, 11036, R1038, R1042,K1052, K1055, K1087, R1090, K1095, N1103, K1108, K1115, K1139, K1158,R1172, K1188, K1276, R1293, A1319, K1340, K1349, and/or K1356 withreference to amino acid position numbering of MbCpf1 (Moraxella bovoculi237).

In any of the non-naturally-occurring CRISPR enzymes:

a single mismatch may exist between the target and a correspondingsequence of the one or more off-target loci; and/or

two, three or four or more mismatches may exist between the target and acorresponding sequence of the one or more off-target loci, and/or

-   -   wherein in (ii) said two, three or four or more mismatches are        contiguous.

In any of the non-naturally-occurring CRISPR enzymes the enzyme in theCRISPR complex may have reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme and wherein theenzyme in the CRISPR complex has increased capability of modifying thesaid target loci as compared to an unmodified enzyme.

In any of the non-naturally-occurring CRISPR enzymes, when in the CRISPRcomplex the relative difference of the modifying capability of theenzyme as between target and at least one off-target locus may beincreased compared to the relative difference of an unmodified enzyme.

In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzymemay comprise one or more additional mutations, wherein the one or moreadditional mutations are in one or more catalytically active domains.

In such non-naturally-occurring CRISPR enzymes, the CRISPR enzyme mayhave reduced or abolished nuclease activity compared with an enzymelacking said one or more additional mutations.

In some such non-naturally-occurring CRISPR enzymes, the CRISPR enzymedoes not direct cleavage of one or other DNA strand at the location ofthe target sequence.

Where the CRISPR enzyme comprises one or more additional mutations inone or more catalytically active domains, the one or more additionalmutations may be in a catalytically active domain of the CRISPR enzymecomprising RuvCI, RuvCII or RuvCIII.

Without being bound by theory, in an aspect of the invention, themethods and mutations described provide for enhancing conformationalrearrangement of CRISPR enzyme domains (e.g. Cpf1 domains) to positionsthat results in cleavage at on-target sits and avoidance of thoseconformational states at off-target sites. CRISPR enzymes cleave targetDNA in a series of coordinated steps. First, the PAM-interacting domainrecognizes the PAM sequence 5′ of the target DNA. After PAM binding, thefirst 10-12 nucleotides of the target sequence (seed sequence) aresampled for gRNA:DNA complementarity, a process dependent on DNA duplexseparation. If the seed sequence nucleotides complement the gRNA, theremainder of DNA is unwound and the full length of gRNA hybridizes withthe target DNA strand. nt-grooves may stabilize the non-targeted DNAstrand and facilitate unwinding through non-specific interactions withpositive charges of the DNA phosphate backbone. RNA:cDNA and CRISPRenzyme:ncDNA interactions drive DNA unwinding in competition againstcDNA:ncDNA rehybridization. Other CRISPR enzyme domains may affect theconformation of nuclease domains as well, for example linkers connectingdifferent domains. Accordingly, the methods and mutations providedencompass, without limitation, RuvCI, RuvCIII, RuvCIII and linkers.Conformational changes in for instance Cpf1 brought about by target DNAbinding, including seed sequence interaction, and interactions with thetarget and non-target DNA strand determine whether the domains arepositioned to trigger nuclease activity. Thus, the mutations and methodsprovided herein demonstrate and enable modifications that go beyond PAMrecognition and RNA-DNA base pairing.

In an aspect, the invention provides CRISPR nucleases as defined herein,such as Cpf1, that comprise an improved equilibrium towardsconformations associated with cleavage activity when involved inon-target interactions and/or improved equilibrium away fromconformations associated with cleavage activity when involved inoff-target interactions. In one aspect, the invention provides Cas (e.g.Cpf1) nucleases with improved proof-reading function, i.e. a Cas (e.g.Cpf1) nuclease which adopts a conformation comprising nuclease activityat an on-target site, and which conformation has increasedunfavorability at an off-target site. Sternberg et al., Nature527(7576):110-3, doi: 10.1038/nature15544, published online 28 Oct.2015. Epub 2015 Oct. 28, used Förster resonance energy transfer FRET)experiments to detect relative orientations of the Cas (e.g. Cpf1)catalytic domains when associated with on- and off-target DNA, and whichmay be extrapolated to the CRISPR enzymes of the present invention (e.g.Cpf1).

The invention further provides methods and mutations for modulatingnuclease activity and/or specificity using modified guide RNAs. Asdiscussed, on-target nuclease activity can be increased or decreased.Also, off-target nuclease activity can be increased or decreased.Further, there can be increased or decreased specificity as to on-targetactivity vs. off-target activity. Modified guide RNAs include, withoutlimitation, truncated guide RNAs, dead guide RNAs, chemically modifiedguide RNAs, guide RNAs associated with functional domains, modifiedguide RNAs comprising functional domains, modified guide RNAs comprisingaptamers, modified guide RNAs comprising adapter proteins, and guideRNAs comprising added or modified loops. In some embodiments, one ormore functional domains are associated with an dead gRNA (dRNA). In someembodiments, a dRNA complex with the CRISPR enzyme directs generegulation by a functional domain at on gene locus while an gRNA directsDNA cleavage by the CRISPR enzyme at another locus. In some embodiments,dRNAs are selected to maximize selectivity of regulation for a genelocus of interest compared to off-target regulation. In someembodiments, dRNAs are selected to maximize target gene regulation andminimize target cleavage.

For the purposes of the following discussion, reference to a functionaldomain could be a functional domain associated with the CRISPR enzyme ora functional domain associated with the adaptor protein.

In the practice of the invention, loops of the gRNA may be extended,without colliding with the Cas (e.g. Cpf1) protein by the insertion ofdistinct RNA loop(s) or distinct sequence(s) that may recruit adaptorproteins that can bind to the distinct RNA loop(s) or distinctsequence(s). The adaptor proteins may include but are not limited toorthogonal RNA-binding protein/aptamer combinations that exist withinthe diversity of bacteriophage coat proteins. A list of such coatproteins includes, but is not limited to: Qβ, F2, GA, fr, JP501, M12,R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95,TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. These adaptorproteins or orthogonal RNA binding proteins can further recruit effectorproteins or fusions which comprise one or more functional domains. Insome embodiments, the functional domain may be selected from the groupconsisting of: transposase domain, integrase domain, recombinase domain,resolvase domain, invertase domain, protease domain, DNAmethyltransferase domain, DNA hydroxylmethylase domain, DNA demethylasedomain, histone acetylase domain, histone deacetylases domain, nucleasedomain, repressor domain, activator domain, nuclear-localization signaldomains, transcription-regulatory protein (or transcription complexrecruiting) domain, cellular uptake activity associated domain, nucleicacid binding domain, antibody presentation domain, histone modifyingenzymes, recruiter of histone modifying enzymes; inhibitor of histonemodifying enzymes, histone methyltransferase, histone demethylase,histone kinase, histone phosphatase, histone ribosylase, histonederibosylase, histone ubiquitinase, histone deubiquitinase, histonebiotinase and histone tail protease. In some preferred embodiments, thefunctional domain is a transcriptional activation domain, such as,without limitation, VP64, p65, MyoD1, HSF1, RTA, SETT/9 or a histoneacetyltransferase. In some embodiments, the functional domain is atranscription repression domain, preferably KRAB. In some embodiments,the transcription repression domain is SID, or concatemers of SID (egSID4X). In some embodiments, the functional domain is an epigeneticmodifying domain, such that an epigenetic modifying enzyme is provided.In some embodiments, the functional domain is an activation domain,which may be the P65 activation domain. In some embodiments, thefunctional domain is a deaminase, such as a cytidine deaminase. Cytidinedeaminese may be directed to a target nucleic acid to where it directsconversion of cytidine to uridine, resulting in C to T substitutions (Gto A on the complementary strand). In such an embodiment, nucleotidesubstitutions can be effected without DNA cleavage.

In an aspect, the invention also provides methods and mutations formodulating Cas (e.g. Cpf1) binding activity and/or binding specificity.In certain embodiments Cas (e.g. Cpf1) proteins lacking nucleaseactivity are used. In certain embodiments, modified guide RNAs areemployed that promote binding but not nuclease activity of a Cas (e.g.Cpf1) nuclease. In such embodiments, on-target binding can be increasedor decreased. Also, in such embodiments off-target binding can beincreased or decreased. Moreover, there can be increased or decreasedspecificity as to on-target binding vs. off-target binding.

In particular embodiments, a reduction of off-target cleavage is ensuredby destabilizing strand separation, more particularly by introducingmutations in the Cpf1 enzyme decreasing the positive charge in the DNAinteracting regions (as described herein and further exemplified forCas9 by Slaymaker et al. 2016 (Science, 1; 351(6268):84-8). In furtherembodiments, a reduction of off-target cleavage is ensured byintroducing mutations into Cpf1 enzyme which affect the interactionbetween the target strand and the guide RNA sequence, more particularlydisrupting interactions between Cpf1 and the phosphate backbone of thetarget DNA strand in such a way as to retain target specific activitybut reduce off-target activity (as described for Cas9 by Kleinstiver etal. 2016, Nature, 28; 529(7587):490-5). In particular embodiments, theoff-target activity is reduced by way of a modified Cpf1 wherein bothinteraction with target strand and non-target strand are modifiedcompared to wild-type Cpf1.

The methods and mutations which can be employed in various combinationsto increase or decrease activity and/or specificity of on-target vs.off-target activity, or increase or decrease binding and/or specificityof on-target vs. off-target binding, can be used to compensate orenhance mutations or modifications made to promote other effects. Suchmutations or modifications made to promote other effects includemutations or modification to the Cas (e.g. Cpf1) and or mutation ormodification made to a guide RNA. In certain embodiments, the methodsand mutations are used with chemically modified guide RNAs. Examples ofguide RNA chemical modifications include, without limitation,incorporation of 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS),or 2′-O-methyl 3′thioPACE (MSP) at one or more terminal nucleotides.Such chemically modified guide RNAs can comprise increased stability andincreased activity as compared to unmodified guide RNAs, thoughon-target vs. off-target specificity is not predictable. (See, Hendel,2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290, publishedonline 29 Jun. 2015). Chemically modified guide RNAs further include,without limitation, RNAs with phosphorothioate linkages and lockednucleic acid (LNA) nucleotides comprising a methylene bridge between the2′ and 4′ carbons of the ribose ring. The methods and mutations of theinvention are used to modulate Cas (e.g. Cpf1) nuclease activity and/orbinding with chemically modified guide RNAs.

In an aspect, the invention provides methods and mutations formodulating binding and/or binding specificity of Cas (e.g. Cpf1)proteins according to the invention as defined herein comprisingfunctional domains such as nucleases, transcriptional activators,transcriptional repressors, and the like. For example, a Cas (e.g. Cpf1)protein can be made nuclease-null, or having altered or reduced nucleaseactivity by introducing mutations such as for instance Cpf1 mutationsdescribed herein elsewhere, and include for instance D917A, E1006A,E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255Aand N1257A with reference to the amino acid positions in the FnCpf1pRuvC domain; or for instance N580A, N584A, T587A, W609A, D610A, K613A,E614A, D616A, K624A, D625A, K627A and Y629A with reference to theputative second nuclease domain as described herein elsewhere. Nucleasedeficient Cas (e.g. Cpf1) proteins are useful for RNA-guided targetsequence dependent delivery of functional domains. The inventionprovides methods and mutations for modulating binding of Cas (e.g. Cpf1)proteins. In one embodiment, the functional domain comprises VP64,providing an RNA-guided transcription factor. In another embodiment, thefunctional domain comprises Fok I, providing an RNA-guided nucleaseactivity. Mention is made of U.S. Pat. Pub. 2014/0356959, U.S. Pat. Pub.2014/0342456, U.S. Pat. Pub. 2015/0031132, and Mali, P. et al., 2013,Science 339(6121):823-6, doi: 10.1126/science.1232033, published online3 Jan. 2013 and through the teachings herein the invention comprehendsmethods and materials of these documents applied in conjunction with theteachings herein. In certain embodiments, on-target binding isincreased. In certain embodiments, off-target binding is decreased. Incertain embodiments, on-target binding is decreased. In certainembodiments, off-target binding is increased. Accordingly, the inventionalso provides for increasing or decreasing specificity of on-targetbinding vs. off-target binding of functionalized Cas (e.g. Cpf1) bindingproteins.

The use of Cas (e.g. Cpf1) as an RNA-guided binding protein is notlimited to nuclease-null Cas (e.g. Cpf1). Cas (e.g. Cpf1) enzymescomprising nuclease activity can also function as RNA-guided bindingproteins when used with certain guide RNAs. For example short guide RNAsand guide RNAs comprising nucleotides mismatched to the target canpromote RNA directed Cas (e.g. Cpf1) binding to a target sequence withlittle or no target cleavage. (See, e.g., Dahlman, 2015, Nat Biotechnol.33(11):1159-1161, doi: 10.1038/nbt.3390, published online 5 Oct. 2015).In an aspect, the invention provides methods and mutations formodulating binding of Cas (e.g. Cpf1) proteins that comprise nucleaseactivity. In certain embodiments, on-target binding is increased. Incertain embodiments, off-target binding is decreased. In certainembodiments, on-target binding is decreased. In certain embodiments,off-target binding is increased. In certain embodiments, there isincreased or decreased specificity of on-target binding vs. off-targetbinding. In certain embodiments, nuclease activity of guide RNA-Cas(e.g. Cpf1) enzyme is also modulated.

RNA-DNA heteroduplex formation is important for cleavage activity andspecificity throughout the target region, not only the seed regionsequence closest to the PAM. Thus, truncated guide RNAs show reducedcleavage activity and specificity. In an aspect, the invention providesmethod and mutations for increasing activity and specificity of cleavageusing altered guide RNAs.

The invention also demonstrates that modifications of Cas (e.g. Cpf1)nuclease specificity can be made in concert with modifications totargeting range. Cas (e.g. Cpf1) mutants can be designed that haveincreased target specificity as well as accommodating modifications inPAM recognition, for example by choosing mutations that alter PAMspecificity and combining those mutations with nt-groove mutations thatincrease (or if desired, decrease) specificity for on-target sequencesvs. off-target sequences. In one such embodiment, a PI domain residue ismutated to accommodate recognition of a desired PAM sequence while oneor more nt-groove amino acids is mutated to alter target specificity.The Cas (e.g. Cpf1) methods and modifications described herein can beused to counter loss of specificity resulting from alteration of PAMrecognition, enhance gain of specificity resulting from alteration ofPAM recognition, counter gain of specificity resulting from alterationof PAM recognition, or enhance loss of specificity resulting fromalteration of PAM recognition.

The methods and mutations can be used with any Cas (e.g. Cpf1) enzymewith altered PAM recognition. Non-limiting examples of PAMs included areas described herein elsewhere.

In further embodiments, the methods and mutations are used modifiedproteins.

In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzymemay comprise one or more heterologous functional domains.

The one or more heterologous functional domains may comprise one or morenuclear localization signal (NLS) domains. The one or more heterologousfunctional domains may comprise at least two or more NLSs.

The one or more heterologous functional domains may comprise one or moretranscriptional activation domains. A transcriptional activation domainmay comprise VP64.

The one or more heterologous functional domains may comprise one or moretranscriptional repression domains. A transcriptional repression domainmay comprise a KRAB domain or a SID domain.

The one or more heterologous functional domain may comprise one or morenuclease domains. The one or more nuclease domains may comprise Fok1.

The one or more heterologous functional domains may have one or more ofthe following activities: methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,nuclease activity, single-strand RNA cleavage activity, double-strandRNA cleavage activity, single-strand DNA cleavage activity,double-strand DNA cleavage activity and nucleic acid binding activity.

The at least one or more heterologous functional domains may be at ornear the amino-terminus of the enzyme and/or at or near thecarboxy-terminus of the enzyme.

The one or more heterologous functional domains may be fused to theCRISPR enzyme, or tethered to the CRISPR enzyme, or linked to the CRISPRenzyme by a linker moiety.

In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzymemay comprise a CRISPR enzyme from an organism from a genus comprisingFrancisella tularensis 1, Francisella tularensis subsp. novicida,Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens, or Porphyromonas macacae (e.g., aCpf1 of one of these organisms modified as described herein), and mayinclude further mutations or alterations or be a chimeric Cas (e.g.Cpf1).

In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzymemay comprise a chimeric Cas (e.g. Cpf1) enzyme comprising a firstfragment from a first Cas (e.g. Cpf1) ortholog and a second fragmentfrom a second Cas (e.g. Cpf1) ortholog, and the first and second Cas(e.g. Cpf1) orthologs are different. At least one of the first andsecond Cas (e.g. Cpf1) orthologs may comprise a Cas (e.g. Cpf1) from anorganism comprising Francisella tularensis 1, Francisella tularensissubsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC20171, Butyrivibrio proteoclasticus, Peregrinibacteria bacteriumGW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_7, Smithellasp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020,Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxellabovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006,Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonasmacacae.

In any of the non-naturally-occurring CRISPR enzymes, a nucleotidesequence encoding the CRISPR enzyme may be codon optimized forexpression in a eukaryote.

In any of the non-naturally-occurring CRISPR enzymes, the cell may be aeukaryotic cell or a prokaryotic cell; wherein the CRISPR complex isoperable in the cell, and whereby the enzyme of the CRISPR complex hasreduced capability of modifying one or more off-target loci of the cellas compared to an unmodified enzyme and/or whereby the enzyme in theCRISPR complex has increased capability of modifying the one or moretarget loci as compared to an unmodified enzyme.

Accordingly, in an aspect, the invention provides a eukaryotic cellcomprising the engineered CRISPR protein or the system as definedherein.

In certain embodiments, the methods as described herein may compriseproviding a Cas (e.g. Cpf1) transgenic cell in which one or more nucleicacids encoding one or more guide RNAs are provided or introducedoperably connected in the cell with a regulatory element comprising apromoter of one or more gene of interest. As used herein, the term “Castransgenic cell” refers to a cell, such as a eukaryotic cell, in which aCas gene has been genomically integrated. The nature, type, or origin ofthe cell are not particularly limiting according to the presentinvention. Also the way how the Cas transgene is introduced in the cellis may vary and can be any method as is known in the art. In certainembodiments, the Cas transgenic cell is obtained by introducing the Castransgene in an isolated cell. In certain other embodiments, the Castransgenic cell is obtained by isolating cells from a Cas transgenicorganism. By means of example, and without limitation, the Castransgenic cell as referred to herein may be derived from a Castransgenic eukaryote, such as a Cas knock-in eukaryote. Reference ismade to WO 2014/093622 (PCT/US13/74667), incorporated herein byreference. Methods of US Patent Publication Nos. 20120017290 and20110265198 assigned to Sangamo BioSciences, Inc. directed to targetingthe Rosa locus may be modified to utilize the CRISPR Cas system of thepresent invention. Methods of US Patent Publication No. 20130236946assigned to Cellectis directed to targeting the Rosa locus may also bemodified to utilize the CRISPR Cas system of the present invention. Bymeans of further example reference is made to Platt et. al. (Cell;159(2):440-455 (2014)), describing a Cas9 knock-in mouse, which isincorporated herein by reference, and which can be extrapolated to theCRISPR enzymes of the present invention as defined herein. The Castransgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassettethereby rendering Cas expression inducible by Cre recombinase.Alternatively, the Cas transgenic cell may be obtained by introducingthe Cas transgene in an isolated cell. Delivery systems for transgenesare well known in the art. By means of example, the Cas transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus, such as for instance one ormore oncogenic mutations, as for instance and without limitationdescribed in Platt et al. (2014), Chen et al., (2014) or Kumar et al.(2009).

The invention also provides a composition comprising the engineeredCRISPR protein as described herein, such as described in this section.

The invention also provides a non-naturally-occurring, engineeredcomposition comprising a CRISPR-Cas complex comprising any thenon-naturally-occurring CRISPR enzyme described above.

In an aspect, the invention provides in a vector system comprising oneor more vectors, wherein the one or more vectors comprises:

a) a first regulatory element operably linked to a nucleotide sequenceencoding the engineered CRISPR protein as defined herein; and optionally

b) a second regulatory element operably linked to one or more nucleotidesequences encoding one or more nucleic acid molecules comprising a guideRNA comprising a guide sequence, a direct repeat sequence, optionallywherein components (a) and (b) are located on same or different vectors.

The invention also provides a non-naturally-occurring, engineeredcomposition comprising:

a delivery system operably configured to deliver CRISPR-Cas complexcomponents or one or more polynucleotide sequences comprising orencoding said components into a cell, and wherein said CRISPR-Cascomplex is operable in the cell,

CRISPR-Cas complex components or one or more polynucleotide sequencesencoding for transcription and/or translation in the cell the CRISPR-Cascomplex components, comprising:

-   -   (I) the non-naturally-occurring CRISPR enzyme (e.g. engineered        Cpf1) as described herein;    -   (II) CRISPR-Cas guide RNA comprising:    -   the guide sequence, and    -   a direct repeat sequence,    -   wherein the enzyme in the CRISPR complex has reduced capability        of modifying one or more off-target loci as compared to an        unmodified enzyme and/or whereby the enzyme in the CRISPR        complex has increased capability of modifying the one or more        target loci as compared to an unmodified enzyme.

In an aspect, the invention also provides in a system comprising theengineered CRISPR protein as described herein, such as described in thissection.

In any such compositions, the delivery system may comprise a yeastsystem, a lipofection system, a microinjection system, a biolisticsystem, virosomes, liposomes, immunoliposomes, polycations,lipid:nucleic acid conjugates or artificial virions, as defined hereinelsewhere.

In any such compositions, the delivery system may comprise a vectorsystem comprising one or more vectors, and wherein component (II)comprises a first regulatory element operably linked to a polynucleotidesequence which comprises the guide sequence, the direct repeat sequenceand optionally, and wherein component (I) comprises a second regulatoryelement operably linked to a polynucleotide sequence encoding the CRISPRenzyme.

In any such compositions, the delivery system may comprise a vectorsystem comprising one or more vectors, and wherein component (II)comprises a first regulatory element operably linked to the guidesequence and the direct repeat sequence, and wherein component (I)comprises a second regulatory element operably linked to apolynucleotide sequence encoding the CRISPR enzyme.

In any such compositions, the composition may comprise more than oneguide RNA, and each guide RNA has a different target whereby there ismultiplexing.

In any such compositions, the polynucleotide sequence(s) may be on onevector.

The invention also provides an engineered, non-naturally occurringClustered Regularly Interspersed Short Palindromic Repeats(CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprisingone or more vectors comprising:

a) a first regulatory element operably linked to a nucleotide sequenceencoding a non-naturally-occurring CRISPR enzyme of any one of theinventive constructs herein; andb) a second regulatory element operably linked to one or more nucleotidesequences encoding one or more of the guide RNAs, the guide RNAcomprising a guide sequence, a direct repeat sequence, wherein:

components (a) and (b) are located on same or different vectors,

-   -   the CRISPR complex is formed;    -   the guide RNA targets the target polynucleotide loci and the        enzyme alters the polynucleotide loci, and    -   the enzyme in the CRISPR complex has reduced capability of        modifying one or more off-target loci as compared to an        unmodified enzyme and/or whereby the enzyme in the CRISPR        complex has increased capability of modifying the one or more        target loci as compared to an unmodified enzyme.

In such a system, component (II) may comprise a first regulatory elementoperably linked to a polynucleotide sequence which comprises the guidesequence, the direct repeat sequence, and wherein component (II) maycomprise a second regulatory element operably linked to a polynucleotidesequence encoding the CRISPR enzyme. In such a system, where applicablethe guide RNA may comprise a chimeric RNA.

In such a system, component (I) may comprise a first regulatory elementoperably linked to the guide sequence and the direct repeat sequence,and wherein component (II) may comprise a second regulatory elementoperably linked to a polynucleotide sequence encoding the CRISPR enzyme.Such a system may comprise more than one guide RNA, and each guide RNAhas a different target whereby there is multiplexing. Components (a) and(b) may be on the same vector.

In any such systems comprising vectors, the one or more vectors maycomprise one or more viral vectors, such as one or more retrovirus,lentivirus, adenovirus, adeno-associated virus or herpes simplex virus.

In any such systems comprising regulatory elements, at least one of saidregulatory elements may comprise a tissue-specific promoter. Thetissue-specific promoter may direct expression in a mammalian bloodcell, in a mammalian liver cell or in a mammalian eye.

In any of the above-described compositions or systems the direct repeatsequence, may comprise one or more protein-interacting RNA aptamers. Theone or more aptamers may be located in the tetraloop. The one or moreaptamers may be capable of binding MS2 bacteriophage coat protein.

In any of the above-described compositions or systems the cell may aeukaryotic cell or a prokaryotic cell; wherein the CRISPR complex isoperable in the cell, and whereby the enzyme of the CRISPR complex hasreduced capability of modifying one or more off-target loci of the cellas compared to an unmodified enzyme and/or whereby the enzyme in theCRISPR complex has increased capability of modifying the one or moretarget loci as compared to an unmodified enzyme.

The invention also provides a CRISPR complex of any of theabove-described compositions or from any of the above-described systems.

The invention also provides a method of modifying a locus of interest ina cell comprising contacting the cell with any of the herein-describedengineered CRISPR enzymes (e.g. engineered Cpf1), compositions or any ofthe herein-described systems or vector systems, or wherein the cellcomprises any of the herein-described CRISPR complexes present withinthe cell. In such methods the cell may be a prokaryotic or eukaryoticcell, preferably a eukaryotic cell. In such methods, an organism maycomprise the cell. In such methods the organism may not be a human orother animal.

Any such method may be ex vivo or in vitro.

In certain embodiments, a nucleotide sequence encoding at least one ofsaid guide RNA or Cas protein is operably connected in the cell with aregulatory element comprising a promoter of a gene of interest, wherebyexpression of at least one CRISPR-Cas system component is driven by thepromoter of the gene of interest. “operably connected” is intended tomean that the nucleotide sequence encoding the guide RNA and/or the Casis linked to the regulatory element(s) in a manner that allows forexpression of the nucleotide sequence, as also referred to hereinelsewhere. The term “regulatory element” is also described hereinelsewhere. According to the invention, the regulatory element comprisesa promoter of a gene of interest, such as preferably a promoter of anendogenous gene of interest. In certain embodiments, the promoter is atits endogenous genomic location. In such embodiments, the nucleic acidencoding the CRISPR and/or Cas is under transcriptional control of thepromoter of the gene of interest at its native genomic location. Incertain other embodiments, the promoter is provided on a (separate)nucleic acid molecule, such as a vector or plasmid, or otherextrachromosomal nucleic acid, i.e. the promoter is not provided at itsnative genomic location. In certain embodiments, the promoter isgenomically integrated at a non-native genomic location.

Any such method, said modifying may comprise modulating gene expression.Said modulating gene expression may comprise activating gene expressionand/or repressing gene expression. Accordingly, in an aspect, theinvention provides in a method of modulating gene expression, whereinthe method comprises introducing the engineered CRISPR protein or systemas described herein into a cell.

The invention also provides a method of treating a disease, disorder orinfection in an individual in need thereof comprising administering aneffective amount of any of the engineered CRISPR enzymes (e.g.engineered Cpf1), compositions, systems or CRISPR complexes describedherein. The disease, disorder or infection may comprise a viralinfection. The viral infection may be HBV.

The invention also provides the use of any of the engineered CRISPRenzymes (e.g. engineered Cpf1), compositions, systems or CRISPRcomplexes described above for gene or genome editing.

The invention also provides a method of altering the expression of agenomic locus of interest in a mammalian cell comprising contacting thecell with the engineered CRISPR enzymes (e.g. engineered Cpf1),compositions, systems or CRISPR complexes described herein and therebydelivering the CRISPR-Cas (vector) and allowing the CRISPR-Cas complexto form and bind to target, and determining if the expression of thegenomic locus has been altered, such as increased or decreasedexpression, or modification of a gene product.

The invention also provides any of the engineered CRISPR enzymes (e.g.engineered Cpf1), compositions, systems or CRISPR complexes describedabove for use as a therapeutic. The therapeutic may be for gene orgenome editing, or gene therapy.

In certain embodiments the activity of engineered CRISPR enzymes (e.g.engineered Cpf1) as described herein comprises genomic DNA cleavage,optionally resulting in decreased transcription of a gene.

In an aspect, the invention provides in an isolated cell having alteredexpression of a genomic locus from the method s as described herein,wherein the altered expression is in comparison with a cell that has notbeen subjected to the method of altering the expression of the genomiclocus. In a related aspect, the invention provides in a cell lineestablished from such cell.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in agenomic locus of interest of for instance an HSC (hematopoietic stemcell), e.g., wherein the genomic locus of interest is associated with amutation associated with an aberrant protein expression or with adisease condition or state, comprising:

-   -   delivering to an HSC, e.g., via contacting an HSC with a        particle containing, a non-naturally occurring or engineered        composition comprising:        -   I. a CRISPR-Cas system guide RNA (gRNA) polynucleotide            sequence, comprising:            -   (a) a guide sequence capable of hybridizing to a target                sequence in a HSC,            -   (b) a direct repeat sequence, and        -   II. a CRISPR enzyme, optionally comprising at least one or            more nuclear localization sequences,

wherein, the guide sequence directs sequence-specific binding of aCRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridized to the target sequence; and

the method may optionally include also delivering a HDR template, e.g.,via the particle contacting the HSC containing or contacting the HSCwith another particle containing, the HDR template wherein the HDRtemplate provides expression of a normal or less aberrant form of theprotein; wherein “normal” is as to wild type, and “aberrant” can be aprotein expression that gives rise to a condition or disease state; and

optionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in agenomic locus of interest of for instance a HSC, e.g., wherein thegenomic locus of interest is associated with a mutation associated withan aberrant protein expression or with a disease condition or state,comprising: delivering to an HSC, e.g., via contacting an HSC with aparticle containing, a non-naturally occurring or engineered compositioncomprising: I. (a) a guide sequence capable of hybridizing to a targetsequence in a HSC, and (b) at least one or more direct repeat sequences,and II. a CRISPR enzyme optionally having one or more NLSs, and theguide sequence directs sequence-specific binding of a CRISPR complex tothe target sequence, and wherein the CRISPR complex comprises the CRISPRenzyme complexed with the guide sequence that is hybridized to thetarget sequence; and

the method may optionally include also delivering a HDR template, e.g.,via the particle contacting the HSC containing or contacting the HSCwith another particle containing, the HDR template wherein the HDRtemplate provides expression of a normal or less aberrant form of theprotein; wherein “normal” is as to wild type, and “aberrant” can be aprotein expression that gives rise to a condition or disease state; and

optionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism.

The delivery can be of one or more polynucleotides encoding any one ormore or all of the CRISPR-complex, advantageously linked to one or moreregulatory elements for in vivo expression, e.g. via particle(s),containing a vector containing the polynucleotide(s) operably linked tothe regulatory element(s). Any or all of the polynucleotide sequenceencoding a CRISPR enzyme, guide sequence, direct repeat sequence, may beRNA. It will be appreciated that where reference is made to apolynucleotide, which is RNA and is said to ‘comprise’ a feature such adirect repeat sequence, the RNA sequence includes the feature. Where thepolynucleotide is DNA and is said to comprise a feature such a directrepeat sequence, the DNA sequence is or can be transcribed into the RNAincluding the feature at issue. Where the feature is a protein, such asthe CRISPR enzyme, the DNA or RNA sequence referred to is, or can be,translated (and in the case of DNA transcribed first).

In certain embodiments the invention provides a method of modifying anorganism, e.g., mammal including human or a non-human mammal or organismby manipulation of a target sequence in a genomic locus of interest ofan HSC e.g., wherein the genomic locus of interest is associated with amutation associated with an aberrant protein expression or with adisease condition or state, comprising delivering, e.g., via contactingof a non-naturally occurring or engineered composition with the HSC,wherein the composition comprises one or more particles comprisingviral, plasmid or nucleic acid molecule vector(s) (e.g. RNA) operablyencoding a composition for expression thereof, wherein the compositioncomprises: (A) I. a first regulatory element operably linked to aCRISPR-Cas system RNA polynucleotide sequence, wherein thepolynucleotide sequence comprises (a) a guide sequence capable ofhybridizing to a target sequence in a eukaryotic cell, (b) a directrepeat sequence and II. a second regulatory element operably linked toan enzyme-coding sequence encoding a CRISPR enzyme comprising at leastone or more nuclear localization sequences (or optionally at least oneor more nuclear localization sequences as some embodiments can involveno NLS), wherein (a), (b) and (c) are arranged in a 5′ to 3′orientation, wherein components I and II are located on the same ordifferent vectors of the system, wherein when transcribed and the guidesequence directs sequence-specific binding of a CRISPR complex to thetarget sequence, and wherein the CRISPR complex comprises the CRISPRenzyme complexed with the guide sequence that is hybridized to thetarget sequence, or (B) a non-naturally occurring or engineeredcomposition comprising a vector system comprising one or more vectorscomprising I. a first regulatory element operably linked to (a) a guidesequence capable of hybridizing to a target sequence in a eukaryoticcell, and (b) at least one or more direct repeat sequences, II. a secondregulatory element operably linked to an enzyme-coding sequence encodinga CRISPR enzyme, and optionally, where applicable, wherein components I,and II are located on the same or different vectors of the system,wherein when transcribed and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,and wherein the CRISPR complex comprises the CRISPR enzyme complexedwith the guide sequence that is hybridized to the target sequence; themethod may optionally include also delivering a HDR template, e.g., viathe particle contacting the HSC containing or contacting the HSC withanother particle containing, the HDR template wherein the HDR templateprovides expression of a normal or less aberrant form of the protein;wherein “normal” is as to wild type, and “aberrant” can be a proteinexpression that gives rise to a condition or disease state; andoptionally the method may include isolating or obtaining HSC from theorganism or non-human organism, optionally expanding the HSC population,performing contacting of the particle(s) with the HSC to obtain amodified HSC population, optionally expanding the population of modifiedHSCs, and optionally administering modified HSCs to the organism ornon-human organism. In some embodiments, components I, II and III arelocated on the same vector. In other embodiments, components I and IIare located on the same vector, while component III is located onanother vector. In other embodiments, components I and III are locatedon the same vector, while component II is located on another vector. Inother embodiments, components II and III are located on the same vector,while component I is located on another vector. In other embodiments,each of components I, II and III is located on different vectors. Theinvention also provides a viral or plasmid vector system as describedherein.

By manipulation of a target sequence, Applicants also mean theepigenetic manipulation of a target sequence. This may be f thechromatin state of a target sequence, such as by modification of themethylation state of the target sequence (i.e. addition or removal ofmethylation or methylation patterns or CpG islands), histonemodification, increasing or reducing accessibility to the targetsequence, or by promoting 3D folding. It will be appreciated that wherereference is made to a method of modifying an organism or mammalincluding human or a non-human mammal or organism by manipulation of atarget sequence in a genomic locus of interest, this may apply to theorganism (or mammal) as a whole or just a single cell or population ofcells from that organism (if the organism is multicellular). In the caseof humans, for instance, Applicants envisage, inter alia, a single cellor a population of cells and these may preferably be modified ex vivoand then re-introduced. In this case, a biopsy or other tissue orbiological fluid sample may be necessary. Stem cells are alsoparticularly preferred in this regard. But, of course, in vivoembodiments are also envisaged. And the invention is especiallyadvantageous as to HSCs.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by manipulation of a first and a secondtarget sequence on opposite strands of a DNA duplex in a genomic locusof interest in a HSC e.g., wherein the genomic locus of interest isassociated with a mutation associated with an aberrant proteinexpression or with a disease condition or state, comprising delivering,e.g., by contacting HSCs with particle(s) comprising a non-naturallyoccurring or engineered composition comprising:

-   -   I. a first CRISPR-Cas (e.g. Cpf1) system RNA polynucleotide        sequence, wherein the first polynucleotide sequence comprises:        -   (a) a first guide sequence capable of hybridizing to the            first target sequence,        -   (b) a first direct repeat sequence, and    -   II. a second CRISPR-Cas (e.g. Cpf1) system guide RNA        polynucleotide sequence, wherein the second polynucleotide        sequence comprises:        -   (a) a second guide sequence capable of hybridizing to the            second target sequence,        -   (b) a second direct repeat sequence, and    -   III. a polynucleotide sequence encoding a CRISPR enzyme        comprising at least one or more nuclear localization sequences        and comprising one or more mutations, wherein (a), (b) and (c)        are arranged in a 5′ to 3′ orientation; or    -   IV. expression product(s) of one or more of I. to III., e.g.,        the first and the second direct repeat sequence, the CRISPR        enzyme;

wherein when transcribed, the first and the second guide sequencedirects sequence-specific binding of a first and a second CRISPR complexto the first and second target sequences respectively, wherein the firstCRISPR complex comprises the CRISPR enzyme complexed with (1) the firstguide sequence that is hybridized to the first target sequence, whereinthe second CRISPR complex comprises the CRISPR enzyme complexed with (1)the second guide sequence that is hybridized to the second targetsequence, wherein the polynucleotide sequence encoding a CRISPR enzymeis DNA or RNA, and wherein the first guide sequence directs cleavage ofone strand of the DNA duplex near the first target sequence and thesecond guide sequence directs cleavage of the other strand near thesecond target sequence inducing a double strand break, thereby modifyingthe organism or the non-human organism; and the method may optionallyinclude also delivering a HDR template, e.g., via the particlecontacting the HSC containing or contacting the HSC with anotherparticle containing, the HDR template wherein the HDR template providesexpression of a normal or less aberrant form of the protein; wherein“normal” is as to wild type, and “aberrant” can be a protein expressionthat gives rise to a condition or disease state; and optionally themethod may include isolating or obtaining HSC from the organism ornon-human organism, optionally expanding the HSC population, performingcontacting of the particle(s) with the HSC to obtain a modified HSCpopulation, optionally expanding the population of modified HSCs, andoptionally administering modified HSCs to the organism or non-humanorganism. In some methods of the invention any or all of thepolynucleotide sequence encoding the CRISPR enzyme, the first and thesecond guide sequence, the first and the second direct repeat sequence.In further embodiments of the invention the polynucleotides encoding thesequence encoding the CRISPR enzyme, the first and the second guidesequence, the first and the second direct repeat sequence, is/are RNAand are delivered via liposomes, nanoparticles, exosomes, microvesicles,or a gene-gun; but, it is advantageous that the delivery is via aparticle. In certain embodiments of the invention, the first and seconddirect repeat sequence share 100% identity. In some embodiments, thepolynucleotides may be comprised within a vector system comprising oneor more vectors. In preferred embodiments, the first CRISPR enzyme hasone or more mutations such that the enzyme is a complementary strandnicking enzyme, and the second CRISPR enzyme has one or more mutationssuch that the enzyme is a non-complementary strand nicking enzyme.Alternatively the first enzyme may be a non-complementary strand nickingenzyme, and the second enzyme may be a complementary strand nickingenzyme. In preferred methods of the invention the first guide sequencedirecting cleavage of one strand of the DNA duplex near the first targetsequence and the second guide sequence directing cleavage of the otherstrand near the second target sequence results in a 5′ overhang. Inembodiments of the invention the 5′ overhang is at most 200 base pairs,preferably at most 100 base pairs, or more preferably at most 50 basepairs. In embodiments of the invention the 5′ overhang is at least 26base pairs, preferably at least 30 base pairs or more preferably 34-50base pairs.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by manipulation of a first and a secondtarget sequence on opposite strands of a DNA duplex in a genomic locusof interest in for instance a HSC e.g., wherein the genomic locus ofinterest is associated with a mutation associated with an aberrantprotein expression or with a disease condition or state, comprisingdelivering, e.g., by contacting HSCs with particle(s) comprising anon-naturally occurring or engineered composition comprising:

-   -   I. a first regulatory element operably linked to        -   (a) a first guide sequence capable of hybridizing to the            first target sequence, and        -   (b) at least one or more direct repeat sequences,    -   II. a second regulatory element operably linked to        -   (a) a second guide sequence capable of hybridizing to the            second target sequence, and        -   (b) at least one or more direct repeat sequences,    -   III. a third regulatory element operably linked to an        enzyme-coding sequence encoding a CRISPR enzyme (e.g. Cpf1), and    -   V. expression product(s) of one or more of I. to IV., e.g., the        first and the second direct repeat sequence, the CRISPR enzyme;        wherein components I, II, III and IV are located on the same or        different vectors of the system, when transcribed, and the first        and the second guide sequence direct sequence-specific binding        of a first and a second CRISPR complex to the first and second        target sequences respectively, wherein the first CRISPR complex        comprises the CRISPR enzyme complexed with (1) the first guide        sequence that is hybridized to the first target sequence,        wherein the second CRISPR complex comprises the CRISPR enzyme        complexed with the second guide sequence that is hybridized to        the second target sequence, wherein the polynucleotide sequence        encoding a CRISPR enzyme is DNA or RNA, and wherein the first        guide sequence directs cleavage of one strand of the DNA duplex        near the first target sequence and the second guide sequence        directs cleavage of the other strand near the second target        sequence inducing a double strand break, thereby modifying the        organism or the non-human organism; and the method may        optionally include also delivering a HDR template, e.g., via the        particle contacting the HSC containing or contacting the HSC        with another particle containing, the HDR template wherein the        HDR template provides expression of a normal or less aberrant        form of the protein; wherein “normal” is as to wild type, and        “aberrant” can be a protein expression that gives rise to a        condition or disease state; and optionally the method may        include isolating or obtaining HSC from the organism or        non-human organism, optionally expanding the HSC population,        performing contacting of the particle(s) with the HSC to obtain        a modified HSC population, optionally expanding the population        of modified HSCs, and optionally administering modified HSCs to        the organism or non-human organism.

The invention also provides a vector system as described herein. Thesystem may comprise one, two, three or four different vectors.Components I, II, III and IV may thus be located on one, two, three orfour different vectors, and all combinations for possible locations ofthe components are herein envisaged, for example: components I, II, IIIand IV can be located on the same vector; components I, II, III and IVcan each be located on different vectors; components I, II, II I and IVmay be located on a total of two or three different vectors, with allcombinations of locations envisaged, etc. In some methods of theinvention any or all of the polynucleotide sequence encoding the CRISPRenzyme, the first and the second guide sequence, the first and thesecond direct repeat sequence is/are RNA. In further embodiments of theinvention the first and second direct repeat sequence share 100%identity. In preferred embodiments, the first CRISPR enzyme has one ormore mutations such that the enzyme is a complementary strand nickingenzyme, and the second CRISPR enzyme has one or more mutations such thatthe enzyme is a non-complementary strand nicking enzyme. Alternativelythe first enzyme may be a non-complementary strand nicking enzyme, andthe second enzyme may be a complementary strand nicking enzyme. In afurther embodiment of the invention, one or more of the viral vectorsare delivered via liposomes, nanoparticles, exosomes, microvesicles, ora gene-gun; but, particle delivery is advantageous.

In preferred methods of the invention the first guide sequence directingcleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directing cleavage of other strand nearthe second target sequence results in a 5′ overhang. In embodiments ofthe invention the 5′ overhang is at most 200 base pairs, preferably atmost 100 base pairs, or more preferably at most 50 base pairs. Inembodiments of the invention the 5′ overhang is at least 26 base pairs,preferably at least 30 base pairs or more preferably 34-50 base pairs.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest in for instance HSC e.g., wherein the genomiclocus of interest is associated with a mutation associated with anaberrant protein expression or with a disease condition or state, byintroducing into the HSC, e.g., by contacting HSCs with particle(s)comprising, a Cas protein having one or more mutations and two guideRNAs that target a first strand and a second strand of the DNA moleculerespectively in the HSC, whereby the guide RNAs target the DNA moleculeand the Cas protein nicks each of the first strand and the second strandof the DNA molecule, whereby a target in the HSC is altered; and,wherein the Cas protein and the two guide RNAs do not naturally occurtogether and the method may optionally include also delivering a HDRtemplate, e.g., via the particle contacting the HSC containing orcontacting the HSC with another particle containing, the HDR templatewherein the HDR template provides expression of a normal or lessaberrant form of the protein; wherein “normal” is as to wild type, and“aberrant” can be a protein expression that gives rise to a condition ordisease state; and optionally the method may include isolating orobtaining HSC from the organism or non-human organism, optionallyexpanding the HSC population, performing contacting of the particle(s)with the HSC to obtain a modified HSC population, optionally expandingthe population of modified HSCs, and optionally administering modifiedHSCs to the organism or non-human organism. In preferred methods of theinvention the Cas protein nicking each of the first strand and thesecond strand of the DNA molecule results in a 5′ overhang. Inembodiments of the invention the 5′ overhang is at most 200 base pairs,preferably at most 100 base pairs, or more preferably at most 50 basepairs. In embodiments of the invention the 5′ overhang is at least 26base pairs, preferably at least 30 base pairs or more preferably 34-50base pairs. In an aspect of the invention the Cas protein is codonoptimized for expression in a eukaryotic cell, preferably a mammaliancell or a human cell. Aspects of the invention relate to the expressionof a gene product being decreased or a template polynucleotide beingfurther introduced into the DNA molecule encoding the gene product or anintervening sequence being excised precisely by allowing the two 5′overhangs to reanneal and ligate or the activity or function of the geneproduct being altered or the expression of the gene product beingincreased. In an embodiment of the invention, the gene product is aprotein.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest in for instance HSC e.g., wherein the genomiclocus of interest is associated with a mutation associated with anaberrant protein expression or with a disease condition or state, byintroducing into the HSC, e.g., by contacting HSCs with particle(s)comprising,

-   -   a) a first regulatory element operably linked to each of two        CRISPR-Cas system guide RNAs that target a first strand and a        second strand respectively of a double stranded DNA molecule of        the HSC, and    -   b) a second regulatory element operably linked to a Cas (e.g.        Cpf1) protein, or    -   c) expression product(s) of a) or b),        wherein components (a) and (b) are located on same or different        vectors of the system, whereby the guide RNAs target the DNA        molecule of the HSC and the Cas protein nicks each of the first        strand and the second strand of the DNA molecule of the HSC;        and, wherein the Cas protein and the two guide RNAs do not        naturally occur together; and the method may optionally include        also delivering a HDR template, e.g., via the particle        contacting the HSC containing or contacting the HSC with another        particle containing, the HDR template wherein the HDR template        provides expression of a normal or less aberrant form of the        protein; wherein “normal” is as to wild type, and “aberrant” can        be a protein expression that gives rise to a condition or        disease state; and optionally the method may include isolating        or obtaining HSC from the organism or non-human organism,        optionally expanding the HSC population, performing contacting        of the particle(s) with the HSC to obtain a modified HSC        population, optionally expanding the population of modified        HSCs, and optionally administering modified HSCs to the organism        or non-human organism. In aspects of the invention the guide        RNAs may comprise a guide sequence fused to a direct repeat        sequence. Aspects of the invention relate to the expression of a        gene product being decreased or a template polynucleotide being        further introduced into the DNA molecule encoding the gene        product or an intervening sequence being excised precisely by        allowing the two 5′ overhangs to reanneal and ligate or the        activity or function of the gene product being altered or the        expression of the gene product being increased. In an embodiment        of the invention, the gene product is a protein. In preferred        embodiments of the invention the vectors of the system are viral        vectors. In a further embodiment, the vectors of the system are        delivered via liposomes, nanoparticles, exosomes, microvesicles,        or a gene-gun; and particles are preferred. In one aspect, the        invention provides a method of modifying a target polynucleotide        in a HSC. In some embodiments, the method comprises allowing a        CRISPR complex to bind to the target polynucleotide to effect        cleavage of said target polynucleotide thereby modifying the        target polynucleotide, wherein the CRISPR complex comprises a        CRISPR enzyme complexed with a guide sequence hybridized to a        target sequence within said target polynucleotide, wherein said        guide sequence is linked to a direct repeat sequence. In some        embodiments, said cleavage comprises cleaving one or two strands        at the location of the target sequence by said CRISPR enzyme. In        some embodiments, said cleavage results in decreased        transcription of a target gene. In some embodiments, the method        further comprises repairing said cleaved target polynucleotide        by homologous recombination with an exogenous template        polynucleotide, wherein said repair results in a mutation        comprising an insertion, deletion, or substitution of one or        more nucleotides of said target polynucleotide. In some        embodiments, said mutation results in one or more amino acid        changes in a protein expressed from a gene comprising the target        sequence. In some embodiments, the method further comprises        delivering one or more vectors or expression product(s) thereof,        e.g., via particle(s), to for instance said HSC, wherein the one        or more vectors drive expression of one or more of: the CRISPR        enzyme, the guide sequence linked to the direct repeat sequence.        In some embodiments, said vectors are delivered to for instance        the HSC in a subject. In some embodiments, said modifying takes        place in said HSC in a cell culture. In some embodiments, the        method further comprises isolating said HSC from a subject prior        to said modifying. In some embodiments, the method further        comprises returning said HSC and/or cells derived therefrom to        said subject.

In one aspect, the invention provides a method of generating forinstance a HSC comprising a mutated disease gene. In some embodiments, adisease gene is any gene associated with an increase in the risk ofhaving or developing a disease. In some embodiments, the methodcomprises (a) introducing one or more vectors or expression product(s)thereof, e.g., via particle(s), into a HSC, wherein the one or morevectors drive expression of one or more of: a CRISPR enzyme, a guidesequence linked to a direct repeat sequence; and (b) allowing a CRISPRcomplex to bind to a target polynucleotide to effect cleavage of thetarget polynucleotide within said disease gene, wherein the CRISPRcomplex comprises the CRISPR enzyme complexed with the guide sequencethat is hybridized to the target sequence within the targetpolynucleotide, and optionally, where applicable, thereby generating aHSC comprising a mutated disease gene. In some embodiments, saidcleavage comprises cleaving one or two strands at the location of thetarget sequence by said CRISPR enzyme. In some embodiments, saidcleavage results in decreased transcription of a target gene. In someembodiments, the method further comprises repairing said cleaved targetpolynucleotide by homologous recombination with an exogenous templatepolynucleotide, wherein said repair results in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of saidtarget polynucleotide. In some embodiments, said mutation results in oneor more amino acid changes in a protein expression from a genecomprising the target sequence. In some embodiments the modified HSC isadministered to an animal to thereby generate an animal model.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in for instance a HSC. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target polynucleotideto effect cleavage of said target polynucleotide thereby modifying thetarget polynucleotide, wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said target polynucleotide, wherein said guide sequence is linkedto a direct repeat sequence. In other embodiments, this inventionprovides a method of modifying expression of a polynucleotide in aeukaryotic cell that arises from for instance an HSC. The methodcomprises increasing or decreasing expression of a target polynucleotideby using a CRISPR complex that binds to the polynucleotide in the HSC;advantageously the CRISPR complex is delivered via particle(s).

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in for instance an HSC. For example,upon the binding of a CRISPR complex to a target sequence in a cell, thetarget polynucleotide is inactivated such that the sequence is nottranscribed, the coded protein is not produced, or the sequence does notfunction as the wild-type sequence does.

In some embodiments the RNA of the CRISPR-Cas system, e.g., the guide orgRNA, can be modified; for instance to include an aptamer or afunctional domain. An aptamer is a synthetic oligonucleotide that bindsto a specific target molecule; for instance a nucleic acid molecule thathas been engineered through repeated rounds of in vitro selection orSELEX (systematic evolution of ligands by exponential enrichment) tobind to various molecular targets such as small molecules, proteins,nucleic acids, and even cells, tissues and organisms. Aptamers areuseful in that they offer molecular recognition properties that rivalthat of antibodies. In addition to their discriminate recognition,aptamers offer advantages over antibodies including that they elicitlittle or no immunogenicity in therapeutic applications. Accordingly, inthe practice of the invention, either or both of the enzyme or the RNAcan include a functional domain.

In some embodiments, the functional domain is a transcriptionalactivation domain, preferably VP64. In some embodiments, the functionaldomain is a transcription repression domain, preferably KRAB. In someembodiments, the transcription repression domain is SID, or concatemersof SID (eg SID4X). In some embodiments, the functional domain is anepigenetic modifying domain, such that an epigenetic modifying enzyme isprovided. In some embodiments, the functional domain is an activationdomain, which may be the P65 activation domain. In some embodiments, thefunctional domain comprises nuclease activity. In one such embodiment,the functional domain comprises Fok1.

The invention also provides an in vitro or ex vivo cell comprising anyof the modified CRISPR enzymes, compositions, systems or complexesdescribed above, or from any of the methods described above. The cellmay be a eukaryotic cell or a prokaryotic cell. The invention alsoprovides progeny of such cells. The invention also provides a product ofany such cell or of any such progeny, wherein the product is a productof the said one or more target loci as modified by the modified CRISPRenzyme of the CRISPR complex. The product may be a peptide, polypeptideor protein. Some such products may be modified by the modified CRISPRenzyme of the CRISPR complex. In some such modified products, theproduct of the target locus is physically distinct from the product ofthe said target locus which has not been modified by the said modifiedCRISPR enzyme.

The invention also provides a polynucleotide molecule comprising apolynucleotide sequence encoding any of the non-naturally-occurringCRISPR enzymes described above.

Any such polynucleotide may further comprise one or more regulatoryelements which are operably linked to the polynucleotide sequenceencoding the non-naturally-occurring CRISPR enzyme.

In any such polynucleotide which comprises one or more regulatoryelements, the one or more regulatory elements may be operably configuredfor expression of the non-naturally-occurring CRISPR enzyme in aeukaryotic cell. The eukaryotic cell may be a human cell. The eukaryoticcell may be a rodent cell, optionally a mouse cell. The eukaryotic cellmay be a yeast cell. The eukaryotic cell may be a chinese hamster ovary(CHO) cell. The eukaryotic cell may be an insect cell.

In any such polynucleotide which comprises one or more regulatoryelements, the one or more regulatory elements may be operably configuredfor expression of the non-naturally-occurring CRISPR enzyme in aprokaryotic cell.

In any such polynucleotide which comprises one or more regulatoryelements, the one or more regulatory elements may operably configuredfor expression of the non-naturally-occurring CRISPR enzyme in an invitro system.

The invention also provides an expression vector comprising any of theabove-described polynucleotide molecules. The invention also providessuch polynucleotide molecule(s), for instance such polynucleotidemolecules operably configured to express the protein and/or the nucleicacid component(s), as well as such vector(s).

The invention further provides for a method of making mutations to a Cas(e.g. Cpf1) or a mutated or modified Cas (e.g. Cpf1) that is an orthologof the CRISPR enzymes according to the invention as described herein,comprising ascertaining amino acid(s) in that ortholog may be in closeproximity or may touch a nucleic acid molecule, e.g., DNA, RNA, gRNA,etc., and/or amino acid(s) analogous or corresponding toherein-identified amino acid(s) in CRISPR enzymes according to theinvention as described herein for modification and/or mutation, andsynthesizing or preparing or expressing the orthologue comprising,consisting of or consisting essentially of modification(s) and/ormutation(s) or mutating as herein-discussed, e.g., modifying, e.g.,changing or mutating, a neutral amino acid to a charged, e.g.,positively charged, amino acid, e.g., from alanine to, e.g., lysine. Theso modified ortholog can be used in CRISPR-Cas systems; and nucleic acidmolecule(s) expressing it may be used in vector or other deliverysystems that deliver molecules or encoding CRISPR-Cas system componentsas herein-discussed.

In an aspect, the invention provides efficient on-target activity andminimizes off target activity. In an aspect, the invention providesefficient on-target cleavage by a CRISPR protein and minimizesoff-target cleavage by the CRISPR protein. In an aspect, the inventionprovides guide specific binding of a CRISPR protein at a gene locuswithout DNA cleavage. In an aspect, the invention provides efficientguide directed on-target binding of a CRISPR protein at a gene locus andminimizes off-target binding of the CRISPR protein. Accordingly, in anaspect, the invention provides target-specific gene regulation. In anaspect, the invention provides guide specific binding of a CRISPR enzymeat a gene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides for cleavage at one gene locus and gene regulation ata different gene locus using a single CRISPR enzyme. In an aspect, theinvention provides orthogonal activation and/or inhibition and/orcleavage of multiple targets using one or more CRISPR protein and/orenzyme.

In another aspect, the present invention provides for a method offunctional screening of genes in a genome in a pool of cells ex vivo orin vivo comprising the administration or expression of a librarycomprising a plurality of CRISPR-Cas system guide RNAs (gRNAs) andwherein the screening further comprises use of a CRISPR enzyme, whereinthe CRISPR complex is modified to comprise a heterologous functionaldomain. In an aspect the invention provides a method for screening agenome comprising the administration to a host or expression in a hostin vivo of a library. In an aspect the invention provides a method asherein discussed further comprising an activator administered to thehost or expressed in the host. In an aspect the invention provides amethod as herein discussed wherein the activator is attached to a CRISPRprotein. In an aspect the invention provides a method as hereindiscussed wherein the activator is attached to the N terminus or the Cterminus of the CRISPR protein. In an aspect the invention provides amethod as herein discussed wherein the activator is attached to a gRNAloop. In an aspect the invention provides a method as herein discussedfurther comprising a repressor administered to the host or expressed inthe host. In an aspect the invention provides a method as hereindiscussed wherein the screening comprises affecting and detecting geneactivation, gene inhibition, or cleavage in the locus.

In an aspect the invention provides a method as herein discussed whereinthe host is a eukaryotic cell. In an aspect the invention provides amethod as herein discussed wherein the host is a mammalian cell. In anaspect the invention provides a method as herein discussed, wherein thehost is a non-human eukaryote cell. In an aspect the invention providesa method as herein discussed, wherein the non-human eukaryote cell is anon-human mammal cell. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal cell may be including,but not limited to, primate bovine, ovine, procine, canine, rodent,Leporidae such as monkey, cow, sheep, pig, dog, rabbit, rat or mousecell. In an aspect the invention provides a method as herein discussed,the cell may be a non-mammalian eukaryotic cell such as poultry bird(e.g., chicken), vertebrate fish (e.g., salmon) or shellfish (e.g.,oyster, claim, lobster, shrimp) cell. In an aspect the inventionprovides a method as herein discussed, the non-human eukaryote cell is aplant cell. The plant cell may be of a monocot or dicot or of a crop orgrain plant such as cassava, corn, sorghum, soybean, wheat, oat or rice.The plant cell may also be of an algae, tree or production plant, fruitor vegetable (e.g., trees such as citrus trees, e.g., orange, grapefruitor lemon trees; peach or nectarine trees; apple or pear trees; nut treessuch as almond or walnut or pistachio trees; nightshade plants; plantsof the genus Brassica; plants of the genus Lactuca; plants of the genusSpinacia; plants of the genus Capsicum; cotton, tobacco, asparagus,carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper,lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape,coffee, cocoa, etc).

In an aspect the invention provides a method as herein discussedcomprising the delivery of the CRISPR-Cas complexes or component(s)thereof or nucleic acid molecule(s) coding therefor, wherein saidnucleic acid molecule(s) are operatively linked to regulatorysequence(s) and expressed in vivo. In an aspect the invention provides amethod as herein discussed wherein the expressing in vivo is via alentivirus, an adenovirus, or an AAV. In an aspect the inventionprovides a method as herein discussed wherein the delivery is via aparticle, a nanoparticle, a lipid or a cell penetrating peptide (CPP).

In particular embodiments it can be of interest to target the CRISPR-Cascomplex to the chloroplast. In many cases, this targeting may beachieved by the presence of an N-terminal extension, called achloroplast transit peptide (CTP) or plastid transit peptide.Chromosomal transgenes from bacterial sources must have a sequenceencoding a CTP sequence fused to a sequence encoding an expressedpolypeptide if the expressed polypeptide is to be compartmentalized inthe plant plastid (e.g. chloroplast). Accordingly, localization of anexogenous polypeptide to a chloroplast is often 1 accomplished by meansof operably linking a polynucleotide sequence encoding a CTP sequence tothe 5′ region of a polynucleotide encoding the exogenous polypeptide.The CTP is removed in a processing step during translocation into theplastid. Processing efficiency may, however, be affected by the aminoacid sequence of the CTP and nearby sequences at the NH 2 terminus ofthe peptide. Other options for targeting to the chloroplast which havebeen described are the maize cab-m7 signal sequence (U.S. Pat. No.7,022,896, WO 97/41228) a pea glutathione reductase signal sequence (WO97/41228) and the CTP described in US2009029861.

In an aspect the invention provides a pair of CRISPR-Cas complexes, eachcomprising a guide RNA (gRNA) comprising a guide sequence capable ofhybridizing to a target sequence in a genomic locus of interest in acell, wherein at least one loop of each sgRNA is modified by theinsertion of distinct RNA sequence(s) that bind to one or more adaptorproteins, and wherein the adaptor protein is associated with one or morefunctional domains, wherein each gRNA of each CRISPR-Cas comprises afunctional domain having a DNA cleavage activity. In an aspect theinvention provides a paired CRISPR-Cas complexes as herein-discussed,wherein the DNA cleavage activity is due to a Fok1 nuclease.

In an aspect the invention provides a method for cutting a targetsequence in a genomic locus of interest comprising delivery to a cell ofthe CRISPR-Cas complexes or component(s) thereof or nucleic acidmolecule(s) coding therefor, wherein said nucleic acid molecule(s) areoperatively linked to regulatory sequence(s) and expressed in vivo. Inan aspect the invention provides a method as herein-discussed whereinthe delivery is via a lentivirus, an adenovirus, or an AAV. In an aspectthe invention provides a method as herein-discussed or paired CRISPR-Cascomplexes as herein-discussed wherein the target sequence for a firstcomplex of the pair is on a first strand of double stranded DNA and thetarget sequence for a second complex of the pair is on a second strandof double stranded DNA. In an aspect the invention provides a method asherein-discussed or paired CRISPR-Cas complexes as herein-discussedwherein the target sequences of the first and second complexes are inproximity to each other such that the DNA is cut in a manner thatfacilitates homology directed repair. In an aspect a herein method canfurther include introducing into the cell template DNA. In an aspect aherein method or herein paired CRISPR-Cas complexes can involve whereineach CRISPR-Cas complex has a CRISPR enzyme that is mutated such that ithas no more than about 5% of the nuclease activity of the CRISPR enzymethat is not mutated.

In an aspect the invention provides a library, method or complex asherein-discussed wherein the gRNA is modified to have at least onenon-coding functional loop, e.g., wherein the at least one non-codingfunctional loop is repressive; for instance, wherein the at least onenon-coding functional loop comprises Alu.

In one aspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring CRISPR-Cas systemcomprising a Cas protein and guide RNA that targets the DNA molecule,whereby the guide RNA targets the DNA molecule encoding the gene productand the Cas protein cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the Casprotein and the guide RNA do not naturally occur together. The inventionfurther comprehends the Cas protein being codon optimized for expressionin a Eukaryotic cell. In a preferred embodiment the Eukaryotic cell is amammalian cell and in a more preferred embodiment the mammalian cell isa human cell. In a further embodiment of the invention, the expressionof the gene product is decreased.

In an aspect, the invention provides altered cells and progeny of thosecells, as well as products made by the cells. CRISPR-Cas (e.g. Cpf1)proteins and systems of the invention are used to produce cellscomprising a modified target locus. In some embodiments, the method maycomprise allowing a nucleic acid-targeting complex to bind to the targetDNA or RNA to effect cleavage of said target DNA or RNA therebymodifying the target DNA or RNA, wherein the nucleic acid-targetingcomplex comprises a nucleic acid-targeting effector protein complexedwith a guide RNA hybridized to a target sequence within said target DNAor RNA. In one aspect, the invention provides a method of repairing agenetic locus in a cell. In another aspect, the invention provides amethod of modifying expression of DNA or RNA in a eukaryotic cell. Insome embodiments, the method comprises allowing a nucleic acid-targetingcomplex to bind to the DNA or RNA such that said binding results inincreased or decreased expression of said DNA or RNA; wherein thenucleic acid-targeting complex comprises a nucleic acid-targetingeffector protein complexed with a guide RNA. Similar considerations andconditions apply as above for methods of modifying a target DNA or RNA.In fact, these sampling, culturing and re-introduction options applyacross the aspects of the present invention. In an aspect, the inventionprovides for methods of modifying a target DNA or RNA in a eukaryoticcell, which may be in vivo, ex vivo or in vitro. In some embodiments,the method comprises sampling a cell or population of cells from a humanor non-human animal, and modifying the cell or cells. Culturing mayoccur at any stage ex vivo. Such cells can be, without limitation, plantcells, animal cells, particular cell types of any organism, includingstem cells, immune cells, T cell, B cells, dendritic cells,cardiovascular cells, epithelial cells, stem cells and the like. Thecells can be modified according to the invention to produce geneproducts, for example in controlled amounts, which may be increased ordecreased, depending on use, and/or mutated. In certain embodiments, agenetic locus of the cell is repaired. The cell or cells may even bere-introduced into the non-human animal or plant. For re-introducedcells it may be preferred that the cells are stem cells.

In an aspect, the invention provides cells which transiently compriseCRISPR systems, or components. For example, CRISPR proteins or enzymesand nucleic acids are transiently provided to a cell and a genetic locusis altered, followed by a decline in the amount of one or morecomponents of the CRISPR system. Subsequently, the cells, progeny of thecells, and organisms which comprise the cells, having acquired a CRISPRmediated genetic alteration, comprise a diminished amount of one or moreCRISPR system components, or no longer contain the one or more CRISPRsystem components. One non-limiting example is a self-inactivatingCRISPR-Cas system such as further described herein. Thus, the inventionprovides cells, and organisms, and progeny of the cells and organismswhich comprise one or more CRISPR-Cas system-altered genetic loci, butessentially lack one or more CRISPR system component. In certainembodiments, the CRISPR system components are substantially absent. Suchcells, tissues and organisms advantageously comprise a desired orselected genetic alteration but have lost CRISPR-Cas components orremnants thereof that potentially might act non-specifically, lead toquestions of safety, or hinder regulatory approval. As well, theinvention provides products made by the cells, organisms, and progeny ofthe cells and organisms.

Inducible Cpf1 CRISPR-Cas Systems (“Split-Cpf1”)

In an aspect the invention provides a non-naturally occurring orengineered inducible Cpf1 CRISPR-Cas system, comprising:

a first Cpf1 fusion construct attached to a first half of an inducibledimer anda second Cpf1 fusion construct attached to a second half of theinducible dimer,

wherein the first Cpf1 fusion construct is operably linked to one ormore nuclear localization signals,

wherein the second Cpf1 fusion construct is operably linked to one ormore nuclear export signals,

wherein contact with an inducer energy source brings the first andsecond halves of the inducible dimer together,

wherein bringing the first and second halves of the inducible dimertogether allows the first and second Cpf1 fusion constructs toconstitute a functional Cpf1 CRISPR-Cas system,

wherein the Cpf1 CRISPR-Cas system comprises a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, and

wherein the functional Cpf1 CRISPR-Cas system binds to the targetsequence and, optionally, edits the genomic locus to alter geneexpression.

In an aspect of the invention in the inducible Cpf1 CRISPR-Cas system,the inducible dimer is or comprises or consists essentially of orconsists of an inducible heterodimer. In an aspect, in inducible Cpf1CRISPR-Cas system, the first half or a first portion or a first fragmentof the inducible heterodimer is or comprises or consists of or consistsessentially of an FKBP, optionally FKBP12. In an aspect of theinvention, in the inducible Cpf1 CRISPR-Cas system, the second half or asecond portion or a second fragment of the inducible heterodimer is orcomprises or consists of or consists essentially of FRB. In an aspect ofthe invention, in the inducible Cpf1 CRISPR-Cas system, the arrangementof the first Cpf1 fusion construct is or comprises or consists of orconsists essentially of N′ terminal Cpf1 part-FRB-NES. In an aspect ofthe invention, in the inducible Cpf1 CRISPR-Cas system, the arrangementof the first Cpf1 fusion construct is or comprises or consists of orconsists essentially of NES-N′ terminal Cpf1 part-FRB-NES. In an aspectof the invention, in the inducible Cpf1 CRISPR-Cas system, thearrangement of the second Cpf1 fusion construct is or comprises orconsists essentially of or consists of C′ terminal Cpf1 part-FKBP-NLS.In an aspect the invention provides in the inducible Cpf1 CRISPR-Cassystem, the arrangement of the second Cpf1 fusion construct is orcomprises or consists of or consists essentially of NLS-C′ terminal Cpf1part-FKBP-NLS. In an aspect, in inducible Cpf1 CRISPR-Cas system therecan be a linker that separates the Cpf1 part from the half or portion orfragment of the inducible dimer. In an aspect, in the inducible Cpf1CRISPR-Cas system, the inducer energy source is or comprises or consistsessentially of or consists of rapamycin. In an aspect, in inducible Cpf1CRISPR-Cas system, the inducible dimer is an inducible homodimer. In anaspect, in inducible Cpf1 CRISPR-Cas system, the Cpf1 is FnCpf1. In anaspect, in the inducible Cpf1 CRISPR-Cas system, one or more functionaldomains are associated with one or both parts of the Cpf1, e.g., thefunctional domains optionally including a transcriptional activator, atranscriptional or a nuclease such as a Fok1 nuclease. In an aspect, inthe inducible Cpf1 CRISPR-Cas system, the functional Cpf1 CRISPR-Cassystem binds to the target sequence and the enzyme is a dead-Cpf1,optionally having a diminished nuclease activity of at least 97%, or100% (or no more than 3% and advantageously 0% nuclease activity) ascompared with the Cpf1 not having the at least one mutation. Theinvention further comprehends and an aspect of the invention provides, apolynucleotide encoding the inducible Cpf1 CRISPR-Cas system as hereindiscussed.

In an aspect, the invention provides a vector for delivery of the firstCpf1 fusion construct, attached to a first half or portion or fragmentof an inducible dimer and operably linked to one or more nuclearlocalization signals, according as herein discussed. In an aspect, theinvention provides a vector for delivery of the second Cpf1 fusionconstruct, attached to a second half or portion or fragment of aninducible dimer and operably linked to one or more nuclear exportsignals.

In an aspect, the invention provides a vector for delivery of both: thefirst Cpf1 fusion construct, attached to a first half or portion orfragment of an inducible dimer and operably linked to one or morenuclear localization signals, as herein discussed; and the second Cpf1fusion construct, attached to a second half or portion or fragment of aninducible dimer and operably linked to one or more nuclear exportsignals, as herein discussed.

In an aspect, the vector can be single plasmid or expression cassette.

The invention, in an aspect, provides a eukaryotic host cell or cellline transformed with any of the vectors herein discussed or expressingthe inducible Cpf1 CRISPR-Cas system as herein discussed.

The invention, in an aspect provides, a transgenic organism transformedwith any of the vectors herein discussed or expressing the inducibleCpf1 CRISPR-Cas system herein discussed, or the progeny thereof. In anaspect, the invention provides a model organism which constitutivelyexpresses the inducible Cpf1 CRISPR-Cas system as herein discussed.

In an aspect, the invention provides non-naturally occurring orengineered inducible Cpf1 CRISPR-Cas system, comprising:

a first Cpf1 fusion construct attached to a first half of an inducibleheterodimer anda second Cpf1 fusion construct attached to a second half of theinducible heterodimer,

wherein the first Cpf1 fusion construct is operably linked to one ormore nuclear localization signals,

wherein the second CPf1 fusion construct is operably linked to a nuclearexport signal,

wherein contact with an inducer energy source brings the first andsecond halves of the inducible heterodimer together,

wherein bringing the first and second halves of the inducibleheterodimer together allows the first and second Cpf1 fusion constructsto constitute a functional Cpf1 CRISPR-Cas system,

wherein the Cpf1 CRISPR-Cas system comprises a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, and

wherein the functional Cpf1 CRISPR-Cas system edits the genomic locus toalter gene expression.

In an aspect, the invention provides a method of treating a subject inneed thereof, comprising inducing gene editing by transforming thesubject with the polynucleotide as herein discussed or any of thevectors herein discussed and administering an inducer energy source tothe subject. The invention comprehends uses of such a polynucleotide orvector in the manufacture of a medicament, e.g., such a medicament fortreating a subject or for such a method of treating a subject. Theinvention comprehends the polynucleotide as herein discussed or any ofthe vectors herein discussed for use in a method of treating a subjectin need thereof comprising inducing gene editing, wherein the methodfurther comprises administering an inducer energy source to the subject.In an aspect, in the method, a repair template is also provided, forexample delivered by a vector comprising said repair template.

The invention also provides a method of treating a subject in needthereof, comprising inducing transcriptional activation or repression bytransforming the subject with the polynucleotide herein discussed or anyof the vectors herein discussed, wherein said polynucleotide or vectorencodes or comprises the catalytically inactive Cpf1 and one or moreassociated functional domains as herein discussed; the method furthercomprising administering an inducer energy source to the subject. Theinvention also provides the polynucleotide herein discussed or any ofthe vectors herein discussed for use in a method of treating a subjectin need thereof comprising inducing transcriptional activation orrepression, wherein the method further comprises administering aninducer energy source to the subject.

Accordingly, the invention comprehends inter alia homodimers as well asheterodimers, dead-Cpf1 or Cpf1 having essentially no nuclease activity,e.g., through mutation, systems or complexes wherein there is one ormore NLS and/or one or more NES; functional domain(s) linked to splitCpf1; methods, including methods of treatment, and uses.

It will be appreciated that where reference is made herein to Cpf1, Cpf1protein or Cpf1 enzyme, this includes the present split Cpf1. In oneaspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring Cpf1 CRISPR-Cas systemcomprising a Cpf1 protein and guide RNA that targets the DNA molecule,whereby the guide RNA targets the DNA molecule encoding the gene productand the Cpf1 protein cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the Cpf1protein and the guide RNA do not naturally occur together. The inventioncomprehends the guide RNA comprising a guide sequence linked to a directrepeat (DR) sequence. The invention further comprehends the Cpf1 proteinbeing codon optimized for expression in a eukaryotic cell. In apreferred embodiment the eukaryotic cell is a mammalian cell and in amore preferred embodiment the mammalian cell is a human cell. In afurther embodiment of the invention, the expression of the gene productis decreased.

In one aspect, the invention provides an engineered, non-naturallyoccurring Cpf1 CRISPR-Cas system comprising a Cpf1 protein and a guideRNA that targets a DNA molecule encoding a gene product in a cell,whereby the guide RNA targets the DNA molecule encoding the gene productand the Cpf1 protein cleaves the DNA molecule encoding the gene product,whereby expression of the gene product is altered; and, wherein the Cpf1protein and the guide RNA do not naturally occur together; thisincluding the present split Cpf1. The invention comprehends the guideRNA comprising a guide sequence linked to a DR sequence. The inventionfurther comprehends the Cpf1 protein being codon optimized forexpression in a eukaryotic cell. In a preferred embodiment theeukaryotic cell is a mammalian cell and in a more preferred embodimentthe mammalian cell is a human cell. In a further embodiment of theinvention, the expression of the gene product is decreased.

In another aspect, the invention provides an engineered, non-naturallyoccurring vector system comprising one or more vectors comprising afirst regulatory element operably linked to a Cpf1 CRISPR-Cas systemguide RNA that targets a DNA molecule encoding a gene product and asecond regulatory element operably linked to a Cpf1 protein; thisincludes the present split Cpf1. Components (a) and (b) may be locatedon same or different vectors of the system. The guide RNA targets theDNA molecule encoding the gene product in a cell and the Cpf1 proteincleaves the DNA molecule encoding the gene product, whereby expressionof the gene product is altered; and, wherein the Cpf1 protein and theguide RNA do not naturally occur together. The invention comprehends theguide RNA comprising a guide sequence linked to a DR sequence. Theinvention further comprehends the Cpf1 protein being codon optimized forexpression in a eukaryotic cell. In a preferred embodiment theeukaryotic cell is a mammalian cell and in a more preferred embodimentthe mammalian cell is a human cell. In a further embodiment of theinvention, the expression of the gene product is decreased.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a DR sequence and one or moreinsertion sites for inserting one or more guide sequences downstream ofthe DR sequence, wherein when expressed, the guide sequence directssequence-specific binding of a Cpf1 CRISPR-Cas complex to a targetsequence in a eukaryotic cell, wherein the Cpf1 CRISPR-Cas complexcomprises Cpf1 complexed with (1) the guide sequence that is hybridizedto the target sequence, and (2) the DR sequence; and (b) a secondregulatory element operably linked to an enzyme-coding sequence encodingsaid Cpf1 enzyme comprising a nuclear localization sequence; whereincomponents (a) and (b) are located on the same or different vectors ofthe system; this includes the present split Cpf1. In some embodiments,component (a) further comprises two or more guide sequences operablylinked to the first regulatory element, wherein when expressed, each ofthe two or more guide sequences direct sequence specific binding of aCpf1 CRISPR-Cas complex to a different target sequence in a eukaryoticcell.

In some embodiments, the Cpf1 CRISPR-Cas complex comprises one or morenuclear localization sequences of sufficient strength to driveaccumulation of said Cpf1 CRISPR-Cas complex in a detectable amount inthe nucleus of a eukaryotic cell. Without wishing to be bound by theory,it is believed that a nuclear localization sequence is not necessary forCpf1 CRISPR-Cas complex activity in eukaryotes, but that including suchsequences enhances activity of the system, especially as to targetingnucleic acid molecules in the nucleus.

In some embodiments, the Cpf1 enzyme is Cpf1 of a bacterial speciesselected from the group consisting of Francisella tularensis 1,Francisella tularensis sub sp. novicida, Prevotella albensis,Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus,Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacteriumGW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6,Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum,Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai,Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3,Prevotella disiens, and Porphyromonas macacae, and may include mutatedCPf1 derived from these organisms. The enzyme may be a Cpf1 homolog orortholog. In some embodiments, the Cpf1 is codon-optimized forexpression in a eukaryotic cell. In some embodiments, the Cpf1 directscleavage of one or two strands at the location of the target sequence.In a preferred embodiment, the strand break is a staggered cut with a 5′overhang. In some embodiments, the first regulatory element is apolymerase III promoter. In some embodiments, the second regulatoryelement is a polymerase II promoter. In some embodiments, the directrepeat has a minimum length of 16 nts and a single stem loop. In furtherembodiments the direct repeat has a length longer than 16 nts,preferably more than 17 nts, and has more than one stem loop oroptimized secondary structures.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a direct repeatsequence and one or more insertion sites for inserting one or more guidesequences downstream of the DR sequence, wherein when expressed, theguide sequence directs sequence-specific binding of a Cpf1 CRISPR-Cascomplex to a target sequence in a eukaryotic cell, wherein the Cpf1CRISPR-Cas complex comprises Cpf1 complexed with (1) the guide sequencethat is hybridized to the target sequence, and (2) the DR sequence;and/or (b) a second regulatory element operably linked to anenzyme-coding sequence encoding said Cpf1 enzyme comprising a nuclearlocalization sequence. In some embodiments, the host cell comprisescomponents (a) and (b); this includes the present split Cpf1. In someembodiments, component (a), component (b), or components (a) and (b) arestably integrated into a genome of the host eukaryotic cell. In someembodiments, component (a) further comprises two or more guide sequencesoperably linked to the first regulatory element, wherein when expressed,each of the two or more guide sequences direct sequence specific bindingof a Cpf1 CRISPR-Cas complex to a different target sequence in aeukaryotic cell. In some embodiments, the CPf1 is codon-optimized forexpression in a eukaryotic cell. In some embodiments, the Cpf1 directscleavage of one or two strands at the location of the target sequence.In a preferred embodiment, the strand break is a staggered cut with a 5′overhang. In some embodiments, the Cpf1 lacks DNA strand cleavageactivity. In some embodiments, the first regulatory element is apolymerase III promoter. In some embodiments, the direct repeat has aminimum length of 16 nts and a single stem loop. In further embodimentsthe direct repeat has a length longer than 16 nts, preferably more than17 nts, and has more than one stem loop or optimized secondarystructures. In an aspect, the invention provides a non-human eukaryoticorganism; preferably a multicellular eukaryotic organism, comprising aeukaryotic host cell according to any of the described embodiments. Inother aspects, the invention provides a eukaryotic organism; preferablya multicellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. The organism in someembodiments of these aspects may be an animal; for example a mammal.Also, the organism may be an arthropod such as an insect. The organismalso may be a plant. Further, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises (a) a first regulatory element operablylinked to a direct repeat sequence and one or more insertion sites forinserting one or more guide sequences downstream of the DR sequence,wherein when expressed, the guide sequence directs sequence-specificbinding of a Cpf1 CRISPR-Cas complex to a target sequence in aeukaryotic cell, wherein the Cpf1 CRISPR-Cas complex comprises Cpf1complexed with (1) the guide sequence that is hybridized to the targetsequence, and (2) the DR sequence; and/or (b) a second regulatoryelement operably linked to an enzyme-coding sequence encoding said Cpf1enzyme comprising a nuclear localization sequence and advantageouslythis includes the present split Cpf1. In some embodiments, the kitcomprises components (a) and (b) located on the same or differentvectors of the system. In some embodiments, component (a) furthercomprises two or more guide sequences operably linked to the firstregulatory element, wherein when expressed, each of the two or moreguide sequences direct sequence specific binding of a Cpf1 CRISPR-Cascomplex to a different target sequence in a eukaryotic cell. In someembodiments, the Cpf1 comprises one or more nuclear localizationsequences of sufficient strength to drive accumulation of said Cpf1 in adetectable amount in the nucleus of a eukaryotic cell. In someembodiments, the Cpf1 enzyme is Cpf1 of a bacterial species selectedfrom the group consisting of Francisella tularensis 1, Francisellatularensis subsp. novicida, Prevotella albensis, Lachnospiraceaebacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteriabacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17,Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceaebacterium MA2020, Candidatus Methanoplasma termitum, Eubacteriumeligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceaebacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, andPorphyromonas macacae, and may include mutated CPf1 derived from theseorganisms. The enzyme may be a Cpf1 homolog or ortholog. In someembodiments, the Cpf1 is codon-optimized for expression in a eukaryoticcell. In some embodiments, the Cpf1 directs cleavage of one or twostrands at the location of the target sequence. In a preferredembodiment, the strand break is a staggered cut with a 5′ overhang. Insome embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.In some embodiments, the direct repeat has a minimum length of 16 ntsand a single stem loop. In further embodiments the direct repeat has alength longer than 16 nts, preferably more than 17 nts, and has morethan one stem loop or optimized secondary structures.

In one aspect, the invention provides a method of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a Cpf1 CRISPR-Cas complex to bind to the targetpolynucleotide to effect cleavage of said target polynucleotide therebymodifying the target polynucleotide, wherein the Cpf1 CRISPR-Cas complexcomprises Cpf1 complexed with a guide sequence hybridized to a targetsequence within said target polynucleotide, wherein said guide sequenceis linked to a direct repeat sequence. In some embodiments, saidcleavage comprises cleaving one or two strands at the location of thetarget sequence by said Cpf1; this includes the present split Cpf1. Insome embodiments, said cleavage results in decreased transcription of atarget gene. In some embodiments, the method further comprises repairingsaid cleaved target polynucleotide by homologous recombination with anexogenous template polynucleotide, wherein said repair results in amutation comprising an insertion, deletion, or substitution of one ormore nucleotides of said target polynucleotide. In some embodiments,said mutation results in one or more amino acid changes in a proteinexpressed from a gene comprising the target sequence. In someembodiments, the method further comprises delivering one or more vectorsto said eukaryotic cell, wherein the one or more vectors driveexpression of one or more of: the Cpf1, and the guide sequence linked tothe DR sequence. In some embodiments, said vectors are delivered to theeukaryotic cell in a subject. In some embodiments, said modifying takesplace in said eukaryotic cell in a cell culture. In some embodiments,the method further comprises isolating said eukaryotic cell from asubject prior to said modifying. In some embodiments, the method furthercomprises returning said eukaryotic cell and/or cells derived therefromto said subject.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a Cpf1 CRISPR-Cas complex to bind to thepolynucleotide such that said binding results in increased or decreasedexpression of said polynucleotide; wherein the Cpf1 CRISPR-Cas complexcomprises Cpf1 complexed with a guide sequence hybridized to a targetsequence within said polynucleotide, wherein said guide sequence islinked to a direct repeat sequence; this includes the present splitCpf1. In some embodiments, the method further comprises delivering oneor more vectors to said eukaryotic cells, wherein the one or morevectors drive expression of one or more of: the Cpf1, and the guidesequence linked to the DR sequence.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated an increase in the risk of havingor developing a disease. In some embodiments, the method comprises (a)introducing one or more vectors into a eukaryotic cell, wherein the oneor more vectors drive expression of one or more of: Cpf1, and a guidesequence linked to a direct repeat sequence; and (b) allowing a Cpf1CRISPR-Cas complex to bind to a target polynucleotide to effect cleavageof the target polynucleotide within said disease gene, wherein the Cpf1CRISPR-Cas complex comprises the Cpf1 complexed with (1) the guidesequence that is hybridized to the target sequence within the targetpolynucleotide, and (2) the DR sequence, thereby generating a modeleukaryotic cell comprising a mutated disease gene; this includes thepresent split Cpf1. In some embodiments, said cleavage comprisescleaving one or two strands at the location of the target sequence bysaid Cpf1. In a preferred embodiment, the strand break is a staggeredcut with a 5′ overhang. In some embodiments, said cleavage results indecreased transcription of a target gene. In some embodiments, themethod further comprises repairing said cleaved target polynucleotide byhomologous recombination with an exogenous template polynucleotide,wherein said repair results in a mutation comprising an insertion,deletion, or substitution of one or more nucleotides of said targetpolynucleotide. In some embodiments, said mutation results in one ormore amino acid changes in a protein expression from a gene comprisingthe target sequence.

In one aspect, the invention provides a method for developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. In some embodiments, a disease gene isany gene associated an increase in the risk of having or developing adisease. In some embodiments, the method comprises (a) contacting a testcompound with a model cell of any one of the described embodiments; and(b) detecting a change in a readout that is indicative of a reduction oran augmentation of a cell signaling event associated with said mutationin said disease gene, thereby developing said biologically active agentthat modulates said cell signaling event associated with said diseasegene.

In one aspect, the invention provides a recombinant polynucleotidecomprising a guide sequence downstream of a direct repeat sequence,wherein the guide sequence when expressed directs sequence-specificbinding of a Cpf1 CRISPR-Cas complex to a corresponding target sequencepresent in a eukaryotic cell. In some embodiments, the target sequenceis a viral sequence present in a eukaryotic cell. In some embodiments,the target sequence is a proto-oncogene or an oncogene.

In one aspect the invention provides for a method of selecting one ormore cell(s) by introducing one or more mutations in a gene in the oneor more cell (s), the method comprising: introducing one or more vectorsinto the cell (s), wherein the one or more vectors drive expression ofone or more of: Cpf1, a guide sequence linked to a direct repeatsequence, and an editing template; wherein the editing templatecomprises the one or more mutations that abolish Cpf1 cleavage; allowinghomologous recombination of the editing template with the targetpolynucleotide in the cell(s) to be selected; allowing a Cpf1 CRISPR-Cascomplex to bind to a target polynucleotide to effect cleavage of thetarget polynucleotide within said gene, wherein the Cpf1 CRISPR-Cascomplex comprises the Cpf1 complexed with (1) the guide sequence that ishybridized to the target sequence within the target polynucleotide, and(2) the direct repeat sequence, wherein binding of the Cpf1 CRISPR-Cascomplex to the target polynucleotide induces cell death, therebyallowing one or more cell(s) in which one or more mutations have beenintroduced to be selected; this includes the present split Cpf1. Inanother preferred embodiment of the invention the cell to be selectedmay be a eukaryotic cell. Aspects of the invention allow for selectionof specific cells without requiring a selection marker or a two-stepprocess that may include a counter-selection system.

Herein there is the phrase “this includes the present split Cpf1” orsimilar text; and, this is to indicate that Cpf1 in embodiments hereincan be a split Cpf1 as herein discussed.

In an aspect the invention involves a non-naturally occurring orengineered inducible Cpf1 CRISPR-Cas system, comprising a first Cpf1fusion construct attached to a first half of an inducible heterodimerand a second Cpf1 fusion construct attached to a second half of theinducible heterodimer, wherein the first CPf1 fusion construct isoperably linked to one or more nuclear localization signals, wherein thesecond CPf1 fusion construct is operably linked to a nuclear exportsignal, wherein contact with an inducer energy source brings the firstand second halves of the inducible heterodimer together, whereinbringing the first and second halves of the inducible heterodimertogether allows the first and second Cpf1 fusion constructs toconstitute a functional Cpf1 CRISPR-Cas system, wherein the Cpf1CRISPR-Cas system comprises a guide RNA (gRNA) comprising a guidesequence capable of hybridizing to a target sequence in a genomic locusof interest in a cell, and wherein the functional Cpf1 CRISPR-Cas systemedits the genomic locus to alter gene expression. In an embodiment ofthe invention the first half of the inducible heterodimer is FKBP12 andthe second half of the inducible heterodimer is FRB. In anotherembodiment of the invention the inducer energy source is rapamycin.

An inducer energy source may be considered to be simply an inducer or adimerizing agent. The term ‘inducer energy source’ is used hereinthroughout for consistency. The inducer energy source (or inducer) actsto reconstitute the Cpf1. In some embodiments, the inducer energy sourcebrings the two parts of the Cpf1 together through the action of the twohalves of the inducible dimer. The two halves of the inducible dimertherefore are brought tougher in the presence of the inducer energysource. The two halves of the dimer will not form into the dimer(dimerize) without the inducer energy source.

Thus, the two halves of the inducible dimer cooperate with the inducerenergy source to dimerize the dimer. This in turn reconstitutes the Cpf1by bringing the first and second parts of the Cpf1 together.

The CRISPR enzyme fusion constructs each comprise one part of the splitCpf1. These are fused, preferably via a linker such as a GlySer linkerdescribed herein, to one of the two halves of the dimer. The two halvesof the dimer may be substantially the same two monomers that togetherthat form the homodimer, or they may be different monomers that togetherform the heterodimer. As such, the two monomers can be thought of as onehalf of the full dimer.

The Cpf1 is split in the sense that the two parts of the Cpf1 enzymesubstantially comprise a functioning Cpf1. That Cpf1 may function as agenome editing enzyme (when forming a complex with the target DNA andthe guide), such as a nickase or a nuclease (cleaving both strands ofthe DNA), or it may be a dead-Cpf1 which is essentially a DNA-bindingprotein with very little or no catalytic activity, due to typicallymutation(s) in its catalytic domains.

The two parts of the split Cpf1 can be thought of as the N′ terminalpart and the C′ terminal part of the split Cpf1. The fusion is typicallyat the split point of the Cpf1. In other words, the C′ terminal of theN′ terminal part of the split Cpf1 is fused to one of the dimer halves,whilst the N′ terminal of the C′ terminal part is fused to the otherdimer half

The Cpf1 does not have to be split in the sense that the break is newlycreated. The split point is typically designed in silico and cloned intothe constructs. Together, the two parts of the split Cpf1, the N′terminal and C′ terminal parts, form a full Cpf1, comprising preferablyat least 70% or more of the wildtype amino acids (or nucleotidesencoding them), preferably at least 80% or more, preferably at least 90%or more, preferably at least 95% or more, and most preferably at least99% or more of the wildtype amino acids (or nucleotides encoding them).Some trimming may be possible, and mutants are envisaged. Non-functionaldomains may be removed entirely. What is important is that the two partsmay be brought together and that the desired Cpf1 function is restoredor reconstituted.

The dimer may be a homodimer or a heterodimer.

One or more, preferably two, NLSs may be used in operable linkage to thefirst Cpf1 construct. One or more, preferably two, NESs may be used inoperable linkage to the first Cpf1 construct. The NLSs and/or the NESspreferably flank the split Cpf1-dimer (i.e., half dimer) fusion, i.e.,one NLS may be positioned at the N′ terminal of the first Cpf1 constructand one NLS may be at the C′ terminal of the first Cpf1 construct.Similarly, one NES may be positioned at the N′ terminal of the secondCpf1 construct and one NES may be at the C′ terminal of the second Cpf1construct. Where reference is made to N′ or C′ terminals, it will beappreciated that these correspond to 5′ ad 3′ ends in the correspondingnucleotide sequence.

A preferred arrangement is that the first Cpf1 construct is arranged5′-NLS-(N′ terminal Cpf1 part)-linker-(first half of the dimer)-NLS-3′.A preferred arrangement is that the second Cpf1 construct is arranged5′-NES-(second half of the dimer)-linker-(C′ terminal Cpf1 part)-NES-3′.A suitable promoter is preferably upstream of each of these constructs.The two constructs may be delivered separately or together.

In some embodiments, one or all of the NES(s) in operable linkage to thesecond CPf1 construct may be swapped out for an NLS. However, this maybe typically not preferred and, in other embodiments, the localizationsignal in operable linkage to the second Cpf1 construct is one or moreNES(s).

It will also be appreciated that the NES may be operably linked to theN′ terminal fragment of the split Cpf1 and that the NLS may be operablylinked to the C′ terminal fragment of the split Cpf1. However, thearrangement where the NLS is operably linked to the N′ terminal fragmentof the split Cpf1 and that the NES is operably linked to the C′ terminalfragment of the split Cpf1 may be preferred.

The NES functions to localize the second Cpf1 fusion construct outsideof the nucleus, at least until the inducer energy source is provided(e.g., at least until an energy source is provided to the inducer toperform its function). The presence of the inducer stimulatesdimerization of the two Cpf1 fusions within the cytoplasm and makes itthermodynamically worthwhile for the dimerized, first and second, Cpf1fusions to localize to the nucleus. Without being bound by theory,Applicants believe that the NES sequesters the second Cpf1 fusion to thecytoplasm (i.e., outside of the nucleus). The NLS on the first Cpf1fusion localizes it to the nucleus. In both cases, Applicants use theNES or NLS to shift an equilibrium (the equilibrium of nucleartransport) to a desired direction. The dimerization typically occursoutside of the nucleus (a very small fraction might happen in thenucleus) and the NLSs on the dimerized complex shift the equilibrium ofnuclear transport to nuclear localization, so the dimerized and hencereconstituted Cpf1 enters the nucleus.

Beneficially, Applicants are able to reconstitute function in the splitCpf1. Transient transfection is used to prove the concept anddimerization occurs in the background in the presence of the inducerenergy source. No activity is seen with separate fragments of the Cpf1.Stable expression through lentiviral delivery is then used to developthis and show that a split Cpf1 approach can be used.

This present split Cpf1 approach is beneficial as it allows the Cpf1activity to be inducible, thus allowing for temporal control.Furthermore, different localization sequences may be used (i.e., the NESand NLS as preferred) to reduce background activity from auto-assembledcomplexes. Tissue specific promoters, for example one for each of thefirst and second Cpf1 fusion constructs, may also be used fortissue-specific targeting, thus providing spatial control. Two differenttissue specific promoters may be used to exert a finer degree of controlif required. The same approach may be used in respect of stage-specificpromoters or there may a mixture of stage and tissue specific promoters,where one of the first and second Cpf1 fusion constructs is under thecontrol of (i.e. operably linked to or comprises) a tissue-specificpromoter, whilst the other of the first and second Cpf1 fusionconstructs is under the control of (i.e. operably linked to orcomprises) a stage-specific promoter.

The inducible Cpf1 CRISPR-Cas system comprises one or more nuclearlocalization sequences (NLSs), as described herein, for example asoperably linked to the first Cpf1 fusion construct. These nuclearlocalization sequences are ideally of sufficient strength to driveaccumulation of said first Cpf1 fusion construct in a detectable amountin the nucleus of a eukaryotic cell. Without wishing to be bound bytheory, it is believed that a nuclear localization sequence is notnecessary for Cpf1 CRISPR-Cas complex activity in eukaryotes, but thatincluding such sequences enhances activity of the system, especially asto targeting nucleic acid molecules in the nucleus, and assists with theoperation of the present 2-part system.

Equally, the second Cpf1 fusion construct is operably linked to anuclear export sequence (NES). Indeed, it may be linked to one or morenuclear export sequences. In other words, the number of export sequencesused with the second Cpf1 fusion construct is preferably 1 or 2 or 3.Typically 2 is preferred, but 1 is enough and so is preferred in someembodiments. Suitable examples of NLS and NES are known in the art. Forexample, a preferred nuclear export signal (NES) is human proteintyrosin kinase 2. Preferred signals will be species specific.

Where the FRB and FKBP system are used, the FKBP is preferably flankedby nuclear localization sequences (NLSs). Where the FRB and FKBP systemare used, the preferred arrangement is N′ terminal Cpf1-FRB-NES:C′terminal Cpf1-FKBP-NLS. Thus, the first Cpf1 fusion construct wouldcomprise the C′ terminal Cpf1 part and the second Cpf1 fusion constructwould comprise the N′ terminal Cpf1 part.

Another beneficial aspect to the present invention is that it may beturned on quickly, i.e. that is has a rapid response. It is believed,without being bound by theory, that Cpf1 activity can be induced throughdimerization of existing (already present) fusion constructs (throughcontact with the inducer energy source) more rapidly than through theexpression (especially translation) of new fusion constructs. As such,the first and second Cpf1 fusion constructs may be expressed in thetarget cell ahead of time, i.e. before Cpf1 activity is required. Cpf1activity can then be temporally controlled and then quickly constitutedthrough addition of the inducer energy source, which ideally acts morequickly (to dimerize the heterodimer and thereby provide Cpf1 activity)than through expression (including induction of transcription) of Cpf1delivered by a vector, for example.

The terms Cpf1 or Cpf1 enzyme and CRISPR enzyme are used interchangeablyherein unless otherwise apparent.

Applicants demonstrate that CPf1 can be split into two components, whichreconstitute a functional nuclease when brought back together. Employingrapamycin sensitive dimerization domains, Applicants generate achemically inducible Cpf1 for temporal control of Cpf1-mediated genomeediting and transcription modulation. Put another way, Applicantsdemonstrate that Cpf1 can be rendered chemically inducible by beingsplit into two fragments and that rapamycin-sensitive dimerizationdomains may be used for controlled reassembly of the Cpf1. Applicantsshow that the re-assembled Cpf1 may be used to mediate genome editing(through nuclease/nickase activity) as well as transcription modulation(as a DNA-binding domain, the so-called “dead Cpf1”).

As such, the use of rapamycin-sensitive dimerization domains ispreferred. Reassembly of the Cpf1 is preferred. Reassembly can bedetermined by restoration of binding activity. Where the Cpf1 is anickase or induces a double-strand break, suitable comparisonpercentages compared to a wildtype are described herein.

Rapamycin treatments can last 12 days. The dose can be 200 nM. Thistemporal and/or molar dosage is an example of an appropriate dose forHuman embryonic kidney 293FT (HEK293FT) cell lines and this may also beused in other cell lines. This figure can be extrapolated out fortherapeutic use in vivo into, for example, mg/kg. However, it is alsoenvisaged that the standard dosage for administering rapamycin to asubject is used here as well. By the “standard dosage”, it is meant thedosage under rapamycin's normal therapeutic use or primary indication(i.e. the dose used when rapamycin is administered for use to preventorgan rejection).

It is noteworthy that the preferred arrangement of Cpf1-FRB/FKBP piecesare separate and inactive until rapamycin-induced dimerization of FRBand FKBP results in reassembly of a functional full-length Cpf1nuclease. Thus, it is preferred that first Cpf1 fusion constructattached to a first half of an inducible heterodimer is deliveredseparately and/or is localized separately from the second Cpf1 fusionconstruct attached to a first half of an inducible heterodimer.

To sequester the Cpf1(N)-FRB fragment in the cytoplasm, where it is lesslikely to dimerize with the nuclear-localized Cpf1(C)-FKBP fragment, itis preferable to use on Cpf1(N)-FRB a single nuclear export sequence(NES) from the human protein tyrosin kinase 2 (Cpf1(N)-FRB-NES). In thepresence of rapamycin, Cpf1(N)-FRB-NES dimerizes with Cpf1(C)-FKBP-2xNLSto reconstitute a complete Cpf1 protein, which shifts the balance ofnuclear trafficking toward nuclear import and allows DNA targeting.

High dosage of Cpf1 can exacerbate indel frequencies at off-target (OT)sequences which exhibit few mismatches to the guide strand. Suchsequences are especially susceptible, if mismatches are non-consecutiveand/or outside of the seed region of the guide. Accordingly, temporalcontrol of Cpf1 activity could be used to reduce dosage in long-termexpression experiments and therefore result in reduced off-target indelscompared to constitutively active Cpf1.

Viral delivery is preferred. In particular, a lentiviral or AAV deliveryvector is envisaged. Applicants generate a split-Cpf1 lentivirusconstruct, similar to the lentiCRISPR plasmid. The split pieces shouldbe small enough to fit the ˜4.7 kb size limitation of AAV.

Applicants demonstrate that stable, low copy expression of split Cpf1can be used to induce substantial indels at a targeted locus withoutsignificant mutation at off-target sites. Applicants clone Cpf1fragments (2 parts based on split 5, described herein).

A dead Cpf1 may also be used, comprising a VP64 transactivation domain,for example added to Cpf1(C)-FKBP-2xNLS (dead-Cpf1(C)-FKBP-2xNLS-VP64).These fragments reconstitute a catalytically inactive Cpf1-VP64 fusion(dead-Cpf1-VP64). Transcriptional activation is induced by VP64 in thepresence of rapamycin to induce the dimerization of the Cpf1(C)-FKBPfusion and the Cpf1(N)-FRB fusion. In other words, Applicants test theinducibility of split dead-Cpf1-VP64 and show that transcriptionalactivation is induced by split dead-Cpf1-VP64 in the presence ofrapamycin. As such, the present inducible Cpf1 may be associated withone or more functional domain, such as a transcriptional activator orrepressor or a nuclease (such as Fok1). A functional domain may be boundto or fused with one part of the split Cpf1.

A preferred arrangement is that the first Cpf1 construct is arranged5′-First Localization Signal-(N′ terminal CPf1 part)-linker-(first halfof the dimer)-First Localization Signal-3′ and the second Cpf1 constructis arranged 5′-Second Localization Signal-(second half of thedimer)-linker-(C′ terminal Cpf1 part)-Second LocalizationSignal-Functional Domain-3′. Here, a functional domain is placed at the3′ end of the second Cpf1 construct. Alternatively, a functional domainmay be placed at the 5′ end of the first Cpf1 construct. One or morefunctional domains may be used at the 3′ end or the 5′ end or at bothends. A suitable promoter is preferably upstream of each of theseconstructs. The two constructs may be delivered separately or together.The Localization Signals may be an NLS or an NES, so long as they arenot inter-mixed on each construct.

In an aspect the invention provides an inducible Cpf1 CRISPR-Cas systemwherein the Cpf1 has a diminished nuclease activity of at least 97%, or100% as compared with the Cpf1 enzyme not having the at least onemutation.

Accordingly, it is also preferred that the Cpf1 is a dead-Cpf1. Ideally,the split should always be so that the catalytic domain(s) areunaffected. For the dead-Cpf1 the intention is that DNA binding occurs,but not cleavage or nickase activity is shown.

In an aspect the invention provides an inducible Cpf1 CRISPR-Cas systemas herein discussed wherein one or more functional domains is associatedwith the Cpf1. This functional domain may be associated with (i.e. boundto or fused with) one part of the split Cpf1 or both. There may be oneassociated with each of the two parts of the split Cpf1. These maytherefore be typically provided as part of the first and/or second Cpf1fusion constructs, as fusions within that construct. The functionaldomains are typically fused via a linker, such as GlySer linker, asdiscussed herein. The one or more functional domains may betranscriptional activation domain or a repressor domain. Although theymay be different domains it is preferred that all the functional domainsare either activator or repressor and that a mixture of the two is notused.

The transcriptional activation domain may comprise VP64, p65, MyoD1,HSF1, RTA or SETT/9.

In an aspect, the invention provides an inducible Cpf1 CRISPR-Cas systemas herein discussed wherein the one or more functional domainsassociated with the Cpf1 is a transcriptional repressor domain.

In an aspect, the invention provides an inducible Cpf1 CRISPR-Cas systemas herein discussed wherein the transcriptional repressor domain is aKRAB domain.

In an aspect, the invention provides an inducible Cpf1 CRISPR-Cas systemas herein discussed wherein the transcriptional repressor domain is aNuE domain, NcoR domain, SID domain or a SID4X domain.

In an aspect the invention provides an inducible Cpf1 CRISPR-Cas systemas herein discussed wherein the one or more functional domainsassociated with the adaptor protein have one or more activitiescomprising methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity, DNA cleavage activity, DNA integration activity or nucleicacid binding activity.

Histone modifying domains are also preferred in some embodiments.Exemplary histone modifying domains are discussed below. Transposasedomains, HR (Homologous Recombination) machinery domains, recombinasedomains, and/or integrase domains are also preferred as the presentfunctional domains. In some embodiments, DNA integration activityincludes HR machinery domains, integrase domains, recombinase domainsand/or transposase domains.

In an aspect the invention provides an inducible Cpf1 CRISPR-Cas systemas herein discussed wherein the DNA cleavage activity is due to anuclease.

In an aspect the invention provides an inducible Cpf1 CRISPR-Cas systemas herein discussed wherein the nuclease comprises a Fok1 nuclease.

The use of such functional domains, which are preferred with the presentsplit Cpf1 system, is also discussed in detail in Konermann et al.(“Genome-scale transcriptional activation with an engineered CRISPR-Cas9complex” Nature published 11 Dec. 2014).

The present system may be used with any guide.

Modified guides may be used in certain embodiments. Particularlypreferred are guides embodying the teachings of Konermann Nature 11 Dec.2014 paper mentioned above. These guides are modified so thatprotein-binding RNA portions (such as aptamers) are added. Suchportion(s) may replace a portion of the guide. Corresponding RNA-bindingprotein domains can be used to then recognise the RNA and recruitfunctional domains, such as those described herein, to the guide. Thisis primarily for use with dead-Cpf1 leading to transcriptionalactivation or repression or DNA cleavage through nucleases such as Fok1.The use of such guides in combination with dead-Cpf1 is powerful, and itis especially powerful if the Cpf1 itself is also associated with itsown functional domain, as discussed herein. When a dead-Cpf1 (with orwithout its own associated functional domain) is induced to reconstitutein accordance with the present invention, i.e. is a split Cpf1, then thetool is especially useful.

A guide RNA (gRNA), also preferred for use in the present invention, cancomprise a guide sequence capable of hybridizing to a target sequence ina genomic locus of interest in a cell, wherein the gRNA is modified bythe insertion of distinct RNA sequence(s) that bind to one or moreadaptor proteins, and wherein the adaptor protein is associated with oneor more functional domains. The Cpf1 may comprise at least one mutation,such that the Cpf1 enzyme has no more than 5% of the nuclease activityof the Cpf1 enzyme not having the at least one mutation; and/or at leastone or more nuclear localization sequences. Also provided is anon-naturally occurring or engineered composition comprising: one ormore guide RNA (gRNA) comprising a guide sequence capable of hybridizingto a target sequence in a genomic locus of interest in a cell, a Cpf1enzyme comprising at least one or more nuclear localization sequences,wherein the CPf1 enzyme comprises at least one mutation, such that theCpf1 enzyme has no more than 5% of the nuclease activity of the Cpf1enzyme not having the at least one mutation, wherein the at least onegRNA is modified by the insertion of distinct RNA sequence(s) that bindto one or more adaptor proteins, and wherein the adaptor protein isassociated with one or more functional domains.

The gRNA that is preferably modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins. The insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins ispreferably an aptamer sequence or two or more aptamer sequences specificto the same or different adaptor protein(s). The adaptor proteinpreferably comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13,JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205,ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. Cell lines stably expressinginter alia split dead-Cpf1 can be useful.

Applicants demonstrate that Cpf1 can be split into two distinctfragments, which reconstitute a functional full-length Cpf1 nucleasewhen brought back together using chemical induction. The split Cpf1architecture will be useful for a variety of applications. For example,split CPf1 may enable genetic strategies for restricting Cpf1 activityto intersectional cell populations by putting each fragment under adifferent tissue specific promoter. Additionally, different chemicallyinducible dimerization domains such as APA and gibberellin may also beemployed.

The inducer energy source is preferably chemical induction.

The split position or location is the point at which the first part ofthe Cpf1 enzyme is separated from the second part. In some embodiments,the first part will comprise or encode amino acids 1 to X, whilst thesecond part will comprise or encode amino acids X+1 to the end. In thisexample, the numbering is contiguous, but this may not always benecessary as amino acids (or the nucleotides encoding them) could betrimmed from the end of either of the split ends, provided thatsufficient DNA binding activity and, if required, DNA nickase orcleavage activity is retained, for example at least 40%, 50%, 60%, 70%,80%, 90% or 95% activity compared to wildtype Cpf1.

The exemplary numbering provided herein may be in reference to thewildtype protein, preferably the wildtype FnCpf1. However, it isenvisaged that mutants of the wildtype Cpf1 such as of FnCpf1 proteincan be used. The numbering may also not follow exactly the FnCpf1numbering as, for instance, some N′ or C′ terminal truncations ordeletions may be used, but this can be addressed using standard sequencealignment tools. Orthologs are also preferred as a sequence alignmenttool.

Thus, the split position may be selected using ordinary skill in theart, for instance based on crystal data and/or computational structurepredictions.

For example, computational analysis of the primary structure of Cpf1nucleases reveals three distinct regions (FIG. 1 ). First a C-terminalRuvC like domain, which is the only functional characterized domain.Second a N-terminal alpha-helical region and thirst a mixed alpha andbeta region, located between the RuvC like domain and the alpha-helicalregion. Several small stretches of unstructured regions are predictedwithin the Cpf1 primary structure. Unstructured regions, which areexposed to the solvent and not conserved within different Cpf1orthologs, may represent preferred sides for splits (FIG. 2 and FIG. 3).

The following table presents non-limiting potential split regions withinAs and LbCpf1. A split site within such a region may be opportune.

Split region AsCpf1 LbCpf1 1 575-588 566-571 2 631-645 754-757 3 653-664— 4 818-844 —

For Fn, As and Lb Cpf1 mutants, it should be readily apparent what thecorresponding position for a potential split site is, for example, basedon a sequence alignment. For non-Fn, As and Lb enzymes one can use thecrystal structure of an ortholog if a relatively high degree of homologyexists between the ortholog and the intended Cpf1, or one can usecomputational prediction.

Ideally, the split position should be located within a region or loop.Preferably, the split position occurs where an interruption of the aminoacid sequence does not result in the partial or full destruction of astructural feature (e.g. alpha-helixes or beta-sheets). Unstructuredregions (regions that do not show up in the crystal structure becausethese regions are not structured enough to be “frozen” in a crystal) areoften preferred options. Applicants can for example make splits inunstructured regions that are exposed on the surface of Cpf1.

Applicants can follow the following procedure which is provided as apreferred example and as guidance. Since unstructured regions don't showup in the crystal structure, Applicants cross-reference the surroundingamino acid sequence of the crystal with the primary amino acid sequenceof the Cpf1. Each unstructured region can be made of for example about 3to 10 amino acids, which does not show up in the crystal. Applicantstherefore make the split in between these amino acids. To include morepotential split sides Applicants include splits located in loops at theoutside of Cpf1 using the same criteria as with unstructured regions.

In some embodiments, the split positon is in an outside loop of theCpf1. In other preferred embodiments, the split position is in anunstructured region of the Cpf1. An unstructured region is typically ahighly flexible outside loop whose structure cannot be readilydetermined from a crystal pattern.

Once the split position has been identified, suitable constructs can bedesigned.

Typically, an NES is positioned at the N′ terminal end of the first partof the split amino acid (or the 5′ end of nucleotide encoding it). Inthat case, an NLS is positioned at the C′ terminal end of the secondpart of the split amino acid (or the 3′ end of the nucleotide encodingit). In this way, the first Cpf1 fusion construct may be operably linkedto one or more nuclear export signals and the second Cpf1 fusionconstruct may be operably linked to a nuclear localization signal.

Of course, the reverse arrangement may be provided, where an NLS ispositioned at the N′ terminal end of the first part of the split aminoacid (or the 5′ end of nucleotide encoding it). In that case, an NES ispositioned at the C′ terminal end of the second part of the split aminoacid (or the 3′ end of the nucleotide encoding it). Thus, the first Cpf1fusion construct may be operably linked to one or more nuclearlocalization signals and the second Cpf1 fusion construct may beoperably linked to a nuclear export signal.

Splits which keep the two parts (either side of the split) roughly thesame length may be advantageous for packing purposes. For example, it isthought to be easier to maintain stoichiometry between both pieces whenthe transcripts are about the same size.

In certain examples, the N- and C-term pieces of human codon-optimizedCpf1 such as FnCpf1 are fused to FRB and FKBP dimerization domains,respectively. This arrangement may be preferred. They may be switchedover (i.e. N′ term to FKBP and C′ term to FRB).

Linkers such as (GGGGS)₃ are preferably used herein to separate the Cpf1fragment from the dimerization domain. (GGGGS)₃ is preferable because itis a relatively long linker (15 amino acids). The glycine residues arethe most flexible and the serine residues enhance the chance that thelinker is on the outside of the protein. (GGGGS)₆ (GGGGS)₉ or (GGGGS)₁₂may preferably be used as alternatives. Other preferred alternatives are(GGGGS)₁, (GGGGS)₂, (GGGGS)₄, (GGGGS)₅, (GGGGS)₇, (GGGGS)₈, (GGGGS)₁₀,or (GGGGS)_(11.)

For example, (GGGGS)₃ may be included between the N′ term Cpf1 fragmentand FRB. For example, (GGGGS)₃ may be included between FKB and the C′term Cpf1 fragment.

Alternative linkers are available, but highly flexible linkers arethought to work best to allow for maximum opportunity for the 2 parts ofthe Cpf1 to come together and thus reconstitute Cpf1 activity. Onealternative is that the NLS of nucleoplasmin can be used as a linker.

A linker can also be used between the Cpf1 and any functional domain.Again, a (GGGGS)₃ linker may be used here (or the 6, 9, or 12 repeatversions therefore) or the NLS of nucleoplasmin can be used as a linkerbetween CPf1 and the functional domain.

Alternatives to the FRB/FKBP system are envisaged. For example the ABAand gibberellin system.

Accordingly, preferred examples of the FKBP family are any one of thefollowing inducible systems. FKBP which dimerizes with CalcineurinA(CNA), in the presence of FK506; FKBP which dimerizes with CyP-Fas, inthe presence of FKCsA; FKBP which dimerizes with FRB, in the presence ofRapamycin; GyrB which dimerizes with GryB, in the presence ofCoumermycin; GAI which dimerizes with GID1, in the presence ofGibberellin; or Snap-tag which dimerizes with HaloTag, in the presenceof HaXS.

Alternatives within the FKBP family itself are also preferred. Forexample, FKBP, which homo-dimerizes (i.e. one FKBP dimerizes withanother FKBP) in the presence of FK1012. Thus, also provided is anon-naturally occurring or engineered inducible Cpf1 CRISPR-Cas system,comprising:

a first Cpf1 fusion construct attached to a first half of an induciblehomoodimer and

a second Cpf1 fusion construct attached to a second half of theinducible homoodimer,

wherein the first Cpf1 fusion construct is operably linked to one ormore nuclear localization signals,

wherein the second Cpf1 fusion construct is operably linked to a(optionally one or more) nuclear export signal(s),

wherein contact with an inducer energy source brings the first andsecond halves of the inducible homoodimer together,

wherein bringing the first and second halves of the inducible homoodimertogether allows the first and second CPf1 fusion constructs toconstitute a functional Cpf1 CRISPR-Cas system,

wherein the Cpf1 CRISPR-Cas system comprises a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, and

wherein the functional Cpf1 CRISPR-Cas system binds to the targetsequence and, optionally, edits the genomic locus to alter geneexpression.

In one embodiment, the homodimer is preferably FKBP and the inducerenergy source is preferably FK1012. In another embodiment, the homodimeris preferably GryB and the inducer energy source is preferablyCoumermycin. In another embodiment, the homodimer is preferably ABA andthe inducer energy source is preferably Gibberellin.

In other embodiments, the dimer is a heterodimer. Preferred examples ofheterodimers are any one of the following inducible systems: FKBP whichdimerizes with CalcineurinA (CNA), in the presence of FK506; FKBP whichdimerizes with CyP-Fas, in the presence of FKCsA; FKBP which dimerizeswith FRB, in the presence of Rapamycin, in the presence of Coumermycin;GAI which dimerizes with GID1, in the presence of Gibberellin; orSnap-tag which dimerizes with HaloTag, in the presence of HaXS.

Applicants used FKBP/FRB because it is well characterized and bothdomains are sufficiently small (<100 amino acids) to assist withpackaging. Furthermore, rapamycin has been used for a long time and sideeffects are well understood. Large dimerization domains (>300 aa) shouldwork too but may require longer linkers to make enable Cpf1reconstitution.

Paulmurugan and Gambhir (Cancer Res, Aug. 15, 2005 65; 7413) discussesthe background to the FRB/FKBP/Rapamycin system. Another useful paper isthe article by Crabtree et al. (Chemistry & Biology 13, 99-107, January2006).

In an example, a single vector, an expression cassette (plasmid) isconstructed. gRNA is under the control of a U6 promoter. Two differentCpf1 splits are used. The split Cpf1 construct is based on a first Cpf1fusion construct, flanked by NLSs, with FKBP fused to C terminal part ofthe split CPf1 via a GlySer linker; and a second CPf1 fusion construct,flanked by NESs, with FRB fused with the N terminal part of the splitCPf1 via a GlySer linker. To separate the first and second Cpf1 fusionconstructs, P2A is used splitting on transcription. The Split Cpf1 showsindel formation similar to wildtype in the presence of rapamycin, butmarkedly lower indel formation than the wildtype in the absence ofrapamycin.

Accordingly, a single vector is provided. The vector comprises:

a first Cpf1 fusion construct attached to a first half of an inducibledimer and

a second Cpf1 fusion construct attached to a second half of theinducible dimer,

wherein the first Cpf1 fusion construct is operably linked to one ormore nuclear localization signals,

wherein the second CPf1 fusion construct is operably linked to one ormore nuclear export signals,

wherein contact with an inducer energy source brings the first andsecond halves of the inducible heterodimer together,

wherein bringing the first and second halves of the inducibleheterodimer together allows the first and second CPf1 fusion constructsto constitute a functional Cpf1 CRISPR-Cas system,

wherein the Cpf1 CRISPR-Cas system comprises a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, and

wherein the functional Cpf1 CRISPR-Cas system binds to the targetsequence and, optionally, edits the genomic locus to alter geneexpression. These elements are preferably provided on a singleconstruct, for example an expression cassette.

The first Cpf1 fusion construct is preferably flanked by at least onenuclear localization signal at each end. The second CPf1 fusionconstruct is preferably flanked by at least one nuclear export signal ateach end.

Also provided is a method of treating a subject in need thereof,comprising inducing gene editing by transforming the subject with thepolynucleotide encoding the system or any of the present vectors andadministering an inducer energy source to the subject. A suitable repairtemplate may also be provided, for example delivered by a vectorcomprising said repair template.

Also provided is a method of treating a subject in need thereof,comprising inducing transcriptional activation or repression bytransforming the subject with the polynucleotide encoding the presentsystem or any of the present vectors, wherein said polynucleotide orvector encodes or comprises the catalytically inactive Cpf1 and one ormore associated functional domains; the method further comprisingadministering an inducer energy source to the subject.

Compositions comprising the present system for use in said method oftreatment are also provided. Use of the present system in themanufacture of a medicament for such methods of treatment are alsoprovided.

Examples of conditions treatable by the present system are describedherein or in documents cited herein.

The single vector can comprise a transcript-splitting agent, for exampleP2A. P2A splits the transcript in two, to separate the first and secondCPf1 fusion constructs. The splitting is due to “ribosomal skipping”. Inessence, the ribosome skips an amino acid during translation, whichbreaks the protein chain and results in two separatepolypeptides/proteins. The single vector is also useful for applicationswhere low background activity is not of concern but a high inducibleactivity is desired.

One example would be the generation of clonal embryonic stem cell lines.The normal procedure is transient transfection with plasmids encoding wtCPf1 or Cpf1 nickases. These plasmids produce Cpf1 molecules, which stayactive for several days and have a higher chance of off target activity.Using the single expression vector for split Cpf1 allows restricting“high” Cpf1 activity to a shorter time window (e.g. one dose of aninducer, such as rapamycin). Without continual (daily) inducer (e.g.rapamycin) treatments the activity of single expression split Cpf1vectors is low and presents a reduced chance of causing unwanted offtarget effects.

A peak of induced Cpf1 activity is beneficial in some embodiments andmay most easily be brought about using a single delivery vector, but itis also possible through a dual vector system (each vector deliveringone half of the split CPf1). The peak may be high activity and for ashort timescale, typically the lifetime of the inducer.

Accordingly, provided is a method for generation of clonal embryonicstem cell lines, comprising transfecting one or more embryonic stemcells with a polynucleotide encoding the present system or one of thepresent vectors to express the present split Cpf1 and administering orcontacting the one or more stem cells with the present inducer energysource to induce reconstitution of the Cpf1. A repair template may beprovided.

As with all methods described herein, it will be appreciated thatsuitable gRNA or guides will be required.

Where functional domains and the like are “associated” with one or otherpart of the enzyme, these are typically fusions. The term “associatedwith” is used here in respect of how one molecule ‘associates’ withrespect to another, for example between parts of the Cpf1 and afunctional domain. In the case of such protein-protein interactions,this association may be viewed in terms of recognition in the way anantibody recognises an epitope. Alternatively, one protein may beassociated with another protein via a fusion of the two, for instanceone subunit being fused to another subunit. Fusion typically occurs byaddition of the amino acid sequence of one to that of the other, forinstance via splicing together of the nucleotide sequences that encodeeach protein or subunit. Alternatively, this may essentially be viewedas binding between two molecules or direct linkage, such as a fusionprotein. In any event, the fusion protein may include a linker betweenthe two subunits of interest (i.e. between the enzyme and the functionaldomain or between the adaptor protein and the functional domain). Thus,in some embodiments, the part of the CPf1 is associated with afunctional domain by binding thereto. In other embodiments, the CPf1 isassociated with a functional domain because the two are fused together,optionally via an intermediate linker. Examples of linkers include theGlySer linkers discussed herein.

Other examples of inducers include light and hormones. For light, theinducible dimers may be heterodimers and include first light-induciblehalf of a dimer and a second (and complimentary) light-inducible half ofa dimer. A preferred example of first and second light-inducible dimerhalves is the CIB1 and CRY2 system. The CIB1 domain is a heterodimericbinding partner of the light-sensitive Cryptochrome 2 (CRY2).

In another example, the blue light-responsive Magnet dimerization system(pMag and nMag) may be fused to the two parts of a split Cpf1 protein.In response to light stimulation, pMag and nMag dimerize and Cpf1reassembles. For example, such system is described in connection withCas9 in Nihongaki et al. (Nat. Biotechnol. 33, 755-790, 2015).

The invention comprehends that the inducer energy source may be heat,ultrasound, electromagnetic energy or chemical. In a preferredembodiment of the invention, the inducer energy source may be anantibiotic, a small molecule, a hormone, a hormone derivative, a steroidor a steroid derivative. In a more preferred embodiment, the inducerenergy source maybe abscisic acid (ABA), doxycycline (DOX), cumate,rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. Theinvention provides that the at least one switch may be selected from thegroup consisting of antibiotic based inducible systems, electromagneticenergy based inducible systems, small molecule based inducible systems,nuclear receptor based inducible systems and hormone based induciblesystems. In a more preferred embodiment the at least one switch may beselected from the group consisting of tetracycline (Tet)/DOX induciblesystems, light inducible systems, ABA inducible systems, cumaterepressor/operator systems, 4OHT/estrogen inducible systems,ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycincomplex) inducible systems. Such inducers are also discussed herein andin PCT/US2013/051418, incorporated herein by reference.

In general, any use that can be made of a Cpf1, whether wt, nickase or adead-Cpf1 (with or without associated functional domains) can be pursuedusing the present split Cpf1 approach. The benefit remains the induciblenature of the Cpf1 activity.

As a further example, split CPf1 fusions with fluorescent proteins likeGFP can be made. This would allow imaging of genomic loci (see “DynamicImaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/CasSystem” Chen B et al. Cell 2013), but in an inducible manner. As such,in some embodiments, one or more of the Cpf1 parts may be associated(and in particular fused with) a fluorescent protein, for example GFP.

Further experiments address whether there is a difference in off-targetcutting, between wild type (wt) and split Cpf1, when on-target cuttingis at the same level. To do this, Applicants use transient transfectionof wt and split Cpf1 plasmids and harvest at different time points.Applicants look for off-target actitation after finding a set of sampleswhere on-target cutting is within +/−5%. Applicants make cell lines withstable expression of wt or split Cpf1 without guides (using lentivirus).After antibiotic selection, guides are delivered with a separatelentivirus and there is harvest at different time points to measureon-/off-target cutting.

Applicants introduce a destabilizing sequence (PEST, see “Use of mRNA-and protein-destabilizing elements to develop a highly responsivereporter system” Voon D C et al. Nucleic Acids Research 2005) into theFRB(N)Cpf1-NES fragment to facilitate faster degradation and thereforereduced stability of the split dead-Cpf1-VP64 complex.

Such destabilizing sequences as described elsewhere in thisspecification (including PEST) can be advantageous for use with splitCpf1 systems.

Cell lines stably expressing split dead-Cpf1-VP64 and MS2-p65-HSF1+guideare generated. A PLX resistance screen can demonstrate that anon-reversible, timed transcriptional activation can be useful in drugscreens. This approach is may be advantageous when a splitdead-Cpf1-VP64 is not reversible.

In one aspect the invention provides a non-naturally occurring orengineered Cpf1 CRISPR-Cas system which may comprise at least one switchwherein the activity of said Cpf1 CRISPR-Cas system is controlled bycontact with at least one inducer energy source as to the switch. In anembodiment of the invention the control as to the at least one switch orthe activity of said Cpf1 CRISPR-Cas system may be activated, enhanced,terminated or repressed. The contact with the at least one inducerenergy source may result in a first effect and a second effect. Thefirst effect may be one or more of nuclear import, nuclear export,recruitment of a secondary component (such as an effector molecule),conformational change (of protein, DNA or RNA), cleavage, release ofcargo (such as a caged molecule or a co-factor), association ordissociation. The second effect may be one or more of activation,enhancement, termination or repression of the control as to the at leastone switch or the activity of said Cpf1 CRISPR-Cas system. In oneembodiment the first effect and the second effect may occur in acascade.

In another aspect of the invention the Cpf1 CRISPR-Cas system mayfurther comprise at least one or more nuclear localization signal (NLS),nuclear export signal (NES), functional domain, flexible linker,mutation, deletion, alteration or truncation. The one or more of theNLS, the NES or the functional domain may be conditionally activated orinactivated. In another embodiment, the mutation may be one or more of amutation in a transcription factor homology region, a mutation in a DNAbinding domain (such as mutating basic residues of a basic helix loophelix), a mutation in an endogenous NLS or a mutation in an endogenousNES. The invention comprehends that the inducer energy source may beheat, ultrasound, electromagnetic energy or chemical. In a preferredembodiment of the invention, the inducer energy source may be anantibiotic, a small molecule, a hormone, a hormone derivative, a steroidor a steroid derivative. In a more preferred embodiment, the inducerenergy source maybe abscisic acid (ABA), doxycycline (DOX), cumate,rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. Theinvention provides that the at least one switch may be selected from thegroup consisting of antibiotic based inducible systems, electromagneticenergy based inducible systems, small molecule based inducible systems,nuclear receptor based inducible systems and hormone based induciblesystems. In a more preferred embodiment the at least one switch may beselected from the group consisting of tetracycline (Tet)/DOX induciblesystems, light inducible systems, ABA inducible systems, cumaterepressor/operator systems, 4OHT/estrogen inducible systems,ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycincomplex) inducible systems.

Aspects of control as detailed in this application relate to at leastone or more switch(es). The term “switch” as used herein refers to asystem or a set of components that act in a coordinated manner to affecta change, encompassing all aspects of biological function such asactivation, repression, enhancement or termination of that function. Inone aspect the term switch encompasses genetic switches which comprisethe basic components of gene regulatory proteins and the specific DNAsequences that these proteins recognize. In one aspect, switches relateto inducible and repressible systems used in gene regulation. Ingeneral, an inducible system may be off unless there is the presence ofsome molecule (called an inducer) that allows for gene expression. Themolecule is said to “induce expression”. The manner by which thishappens is dependent on the control mechanisms as well as differences incell type. A repressible system is on except in the presence of somemolecule (called a corepressor) that suppresses gene expression. Themolecule is said to “repress expression”. The manner by which thishappens is dependent on the control mechanisms as well as differences incell type. The term “inducible” as used herein may encompass all aspectsof a switch irrespective of the molecular mechanism involved.Accordingly a switch as comprehended by the invention may include but isnot limited to antibiotic based inducible systems, electromagneticenergy based inducible systems, small molecule based inducible systems,nuclear receptor based inducible systems and hormone based induciblesystems. In preferred embodiments the switch may be a tetracycline(Tet)/DOX inducible system, a light inducible systems, a Abscisic acid(ABA) inducible system, a cumate repressor/operator system, a4OHT/estrogen inducible system, an ecdysone-based inducible systems or aFKBP12/FRAP (FKBP12-rapamycin complex) inducible system.

The present Cpf1 CRISPR-Cas system may be designed to modulate or alterexpression of individual endogenous genes in a temporally and spatiallyprecise manner. The Cpf1 CRISPR-Cas system may be designed to bind tothe promoter sequence of the gene of interest to change gene expression.The Cpf1 may be spilt into two where one half is fused to one half ofthe cryptochrome heterodimer (cryptochrome-2 or CIB1), while theremaining cryptochrome partner is fused to the other half of the Cpf1.In some aspects, a transcriptional effector domain may also be includedin the Cpf1 CRISPR-Cas system. Effector domains may be eitheractivators, such as VP16, VP64, or p65, or repressors, such as KRAB,EnR, or SID. In unstimulated state, the one half Cpf1-cryptochrome2protein localizes to the promoter of the gene of interest, but is notbound to the CIB1-effector protein. Upon stimulation with blue spectrumlight, cryptochrome-2 becomes activated, undergoes a conformationalchange, and reveals its binding domain. CIB1, in turn, binds tocryptochrome-2 resulting in localization of the second half of the Cpf1to the promoter region of the gene of interest and initiating genomeediting which may result in gene overexpression or silencing. Aspects ofLITEs are further described in Liu, H et al., Science, 2008 and KennedyM et al., Nature Methods 2010, the contents of which are hereinincorporated by reference in their entirety.

Activator and repressor domains which may further modulate function maybe selected on the basis of species, strength, mechanism, duration,size, or any number of other parameters. Preferred effector domainsinclude, but are not limited to, a transposase domain, integrase domain,recombinase domain, resolvase domain, invertase domain, protease domain,DNA methyltransferase domain, DNA demethylase domain, histone acetylasedomain, histone deacetylases domain, nuclease domain, repressor domain,activator domain, nuclear-localization signal domains,transcription-protein recruiting domain, cellular uptake activityassociated domain, nucleic acid binding domain or antibody presentationdomain.

There are several different ways to generate chemical inducible systemsas well: 1. ABI-PYL based system inducible by Abscisic Acid (ABA) (see,e.g., website atstke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/rs2), 2.FKBP-FRB based system inducible by rapamycin (or related chemicals basedon rapamycin) (see, e.g., website atnature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3. GID1-GAI basedsystem inducible by Gibberellin (GA) (see, e.g., website atnature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

Another system contemplated by the present invention is a chemicalinducible system based on change in sub-cellular localization.Applicants also comprehend an inducible Cpf1 CRISPR-Cas systemengineered to target a genomic locus of interest wherein the Cpf1 enzymeis split into two fusion constructs that are further linked to differentparts of a chemical or energy sensitive protein. This chemical or energysensitive protein will lead to a change in the sub-cellular localizationof either half of the CPf1 enzyme (i.e. transportation of either half ofthe Cpf1 enzyme from cytoplasm into the nucleus of the cells) upon thebinding of a chemical or energy transfer to the chemical or energysensitive protein. This transportation of fusion constructs from onesub-cellular compartments or organelles, in which its activity issequestered due to lack of substrate for the reconstituted Cpf1CRISPR-Cas system, into another one in which the substrate is presentwould allow the components to come together and reconstitute functionalactivity and to then come in contact with its desired substrate (i.e.genomic DNA in the mammalian nucleus) and result in activation orrepression of target gene expression.

Other inducible systems are contemplated such as, but not limited to,regulation by heavy-metals [Mayo K E et al., Cell 1982, 29:99-108;Searle P F et al., Mol Cell Biol 1985, 5:1480-1489 and Brinster R L etal., Nature (London) 1982, 296:39-42], steroid hormones [Hynes N E etal., Proc Natl Acad Sci USA 1981, 78:2038-2042; Klock G et al., Nature(London) 1987, 329:734-736 and Lee F et al., Nature (London) 1981,294:228-232.], heat shock [Nouer L: Heat Shock Response. Boca Raton,Fla.: CRC; 1991] and other reagents have been developed [Mullick A,Massie B: Transcription, translation and the control of gene expression.In Encyclopedia of Cell Technology Edited by: Speir R E. Wiley;2000:1140-1164 and Fussenegger M, Biotechnol Prog 2001, 17:1-51].However, there are limitations with these inducible mammalian promoterssuch as “leakiness” of the “off” state and pleiotropic effects ofinducers (heat shock, heavy metals, glucocorticoids etc.). The use ofinsect hormones (ecdysone) has been proposed in an attempt to reduce theinterference with cellular processes in mammalian cells [No D et al.,Proc Natl Acad Sci USA 1996, 93:3346-3351]. Another elegant system usesrapamycin as the inducer [Rivera V M et al., Nat Med 1996, 2:1028-1032]but the role of rapamycin as an immunosuppressant was a major limitationto its use in vivo and therefore it was necessary to find a biologicallyinert compound [Saez E et al., Proc Natl Acad Sci USA 2000,97:14512-14517] for the control of gene expression.

In particular embodiments, the gene editing systems described herein areplaced under the control of a passcode kill switch, which is amechanisms which efficiently kills the host cell when the conditions ofthe cell are altered. This is ensured by introducing hybrid LacI-GalRfamily transcription factors, which require the presence of IPTG to beswitched on (Chan et al. 2015 Nature Nature Chemical Biologydoi:10.1038/nchembio.1979 which can be used to drive a gene encoding anenzyme critical for cell-survival. By combining different transcriptionfactors sensitive to different chemicals, a “code” can be generated,This system can be used to spatially and temporally control the extentof CRISPR-induced genetic modifications, which can be of interest indifferent fields including therapeutic applications and may also be ofinterest to avoid the “escape” of GMOs from their intended environment.

Self-Inactivating Systems

Once all copies of a gene in the genome of a cell have been edited,continued CRISRP/Cpf1 expression in that cell is no longer necessary.Indeed, sustained expression would be undesirable in case of off-targeteffects at unintended genomic sites, etc. Thus time-limited expressionwould be useful. Inducible expression offers one approach, but inaddition Applicants envisage a Self-Inactivating CRISPR-Cpf1 system thatrelies on the use of a non-coding guide target sequence within theCRISPR vector itself. Thus, after expression begins, the CRISPR systemwill lead to its own destruction, but before destruction is complete itwill have time to edit the genomic copies of the target gene (which,with a normal point mutation in a diploid cell, requires at most twoedits). Simply, the self inactivating CRISPR-Cas system includesadditional RNA (i.e., guide RNA) that targets the coding sequence forthe CRISPR enzyme itself or that targets one or more non-coding guidetarget sequences complementary to unique sequences present in one ormore of the following:

(a) within the promoter driving expression of the non-coding RNAelements,

(b) within the promoter driving expression of the Cpf1 gene,

(c) within 100 bp of the ATG translational start codon in the Cpf1coding sequence,

(d) within the inverted terminal repeat (iTR) of a viral deliveryvector, e.g., in the AAV genome.

Furthermore, that RNA can be delivered via a vector, e.g., a separatevector or the same vector that is encoding the CRISPR complex. Whenprovided by a separate vector, the CRISPR RNA that targets Cpf1expression can be administered sequentially or simultaneously. Whenadministered sequentially, the CRISPR RNA that targets Cpf1 expressionis to be delivered after the CRISPR RNA that is intended for e.g. geneediting or gene engineering. This period may be a period of minutes(e.g. 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60minutes). This period may be a period of hours (e.g. 2 hours, 4 hours, 6hours, 8 hours, 12 hours, 24 hours). This period may be a period of days(e.g. 2 days, 3 days, 4 days, 7 days). This period may be a period ofweeks (e.g. 2 weeks, 3 weeks, 4 weeks). This period may be a period ofmonths (e.g. 2 months, 4 months, 8 months, 12 months). This period maybe a period of years (2 years, 3 years, 4 years). In this fashion, theCas enzyme associates with a first gRNA capable of hybridizing to afirst target, such as a genomic locus or loci of interest and undertakesthe function(s) desired of the CRISPR-Cas system (e.g., geneengineering); and subsequently the Cpf1 enzyme may then associate withthe second gRNA capable of hybridizing to the sequence comprising atleast part of the Cpf1 or CRISPR cassette. Where the gRNA targets thesequences encoding expression of the Cpf1 protein, the enzyme becomesimpeded and the system becomes self inactivating. In the same manner,CRISPR RNA that targets Cpf1 expression applied via, for exampleliposome, lipofection, nanoparticles, microvesicles as explained herein,may be administered sequentially or simultaneously. Similarly,self-inactivation may be used for inactivation of one or more guide RNAused to target one or more targets.

In some aspects, a single gRNA is provided that is capable ofhybridization to a sequence downstream of a CRISPR enzyme start codon,whereby after a period of time there is a loss of the CRISPR enzymeexpression. In some aspects, one or more gRNA(s) are provided that arecapable of hybridization to one or more coding or non-coding regions ofthe polynucleotide encoding the CRISPR-Cas system, whereby after aperiod of time there is a inactivation of one or more, or in some casesall, of the CRISPR-Cas systems. In some aspects of the system, and notto be limited by theory, the cell may comprise a plurality of CRISPR-Cascomplexes, wherein a first subset of CRISPR complexes comprise a firstgRNA capable of targeting a genomic locus or loci to be edited, and asecond subset of CRISPR complexes comprise at least one second gRNAcapable of targeting the polynucleotide encoding the CRISPR-Cas system,wherein the first subset of CRISPR-Cas complexes mediate editing of thetargeted genomic locus or loci and the second subset of CRISPR complexeseventually inactivate the CRISPR-Cas system, thereby inactivatingfurther CRISPR-Cas expression in the cell.

Thus the invention provides a CRISPR-Cas system comprising one or morevectors for delivery to a eukaryotic cell, wherein the vector(s)encode(s): (i) a CRISPR enzyme, more particularly Cpf1; (ii) a firstguide RNA capable of hybridizing to a target sequence in the cell; and(iii) a second guide RNA capable of hybridizing to one or more targetsequence(s) in the vector which encodes the CRISPR enzyme, Whenexpressed within the cell, the first guide RNA directs sequence-specificbinding of a first CRISPR complex to the target sequence in the cell;the second guide RNA directs sequence-specific binding of a secondCRISPR complex to the target sequence in the vector which encodes theCRISPR enzyme; the CRISPR complexes comprise a CRISPR enzyme bound to aguide RNA, whereby a guide RNA can hybridize to its target sequence; andthe second CRISPR complex inactivates the CRISPR-Cas system to preventcontinued expression of the CRISPR enzyme by the cell.

Further characteristics of the vector(s), the encoded enzyme, the guidesequences, etc. are disclosed elsewhere herein. The system can encode(i) a CRISPR enzyme, more particularly Cpf1; (ii) a first gRNAcomprising a sequence capable of hybridizing to a first target sequencein the cell, (iii) a second guide RNA capable of hybridizing to thevector which encodes the CRISPR enzyme. Similarly, the enzyme caninclude one or more NLS, etc.

The various coding sequences (CRISPR enzyme, guide RNAs) can be includedon a single vector or on multiple vectors. For instance, it is possibleto encode the enzyme on one vector and the various RNA sequences onanother vector, or to encode the enzyme and one gRNA on one vector, andthe remaining gRNA on another vector, or any other permutation. Ingeneral, a system using a total of one or two different vectors ispreferred.

Where multiple vectors are used, it is possible to deliver them inunequal numbers, and ideally with an excess of a vector which encodesthe first guide RNA relative to the second guide RNA, thereby assistingin delaying final inactivation of the CRISPR system until genome editinghas had a chance to occur.

The first guide RNA can target any target sequence of interest within agenome, as described elsewhere herein. The second guide RNA targets asequence within the vector which encodes the CRISPR Cas9 enzyme, andthereby inactivates the enzyme's expression from that vector. Thus thetarget sequence in the vector must be capable of inactivatingexpression. Suitable target sequences can be, for instance, near to orwithin the translational start codon for the Cpf1 coding sequence, in anon-coding sequence in the promoter driving expression of the non-codingRNA elements, within the promoter driving expression of the Cpf1 gene,within 100 bp of the ATG translational start codon in the Cpf1 codingsequence, and/or within the inverted terminal repeat (iTR) of a viraldelivery vector, e.g., in the AAV genome. A double stranded break nearthis region can induce a frame shift in the Cpf1 coding sequence,causing a loss of protein expression. An alternative target sequence forthe “self-inactivating” guide RNA would aim to edit/inactivateregulatory regions/sequences needed for the expression of theCRISPR-Cpf1 system or for the stability of the vector. For instance, ifthe promoter for the Cpf1 coding sequence is disrupted thentranscription can be inhibited or prevented. Similarly, if a vectorincludes sequences for replication, maintenance or stability then it ispossible to target these. For instance, in a AAV vector a useful targetsequence is within the iTR. Other useful sequences to target can bepromoter sequences, polyadenlyation sites, etc.

Furthermore, if the guide RNAs are expressed in array format, the“self-inactivating” guide RNAs that target both promoters simultaneouslywill result in the excision of the intervening nucleotides from withinthe CRISPR-Cas expression construct, effectively leading to its completeinactivation. Similarly, excision of the intervening nucleotides willresult where the guide RNAs target both ITRs, or targets two or moreother CRISPR-Cas components simultaneously. Self-inactivation asexplained herein is applicable, in general, with CRISPR-Cpf1 systems inorder to provide regulation of the CRISPR-Cpf1. For example,self-inactivation as explained herein may be applied to the CRISPRrepair of mutations, for example expansion disorders, as explainedherein. As a result of this self-inactivation, CRISPR repair is onlytransiently active.

Addition of non-targeting nucleotides to the 5′ end (e.g. 1-10nucleotides, preferably 1-5 nucleotides) of the “self-inactivating”guide RNA can be used to delay its processing and/or modify itsefficiency as a means of ensuring editing at the targeted genomic locusprior to CRISPR-Cpf1 shutdown.

In one aspect of the self-inactivating AAV-CRISPR-Cpf1 system, plasmidsthat co-express one or more gRNA targeting genomic sequences of interest(e.g. 1-2, 1-5, 1-10, 1-15, 1-20, 1-30) may be established with“self-inactivating” gRNAs that target an LbCpf1 sequence at or near theengineered ATG start site (e.g. within 5 nucleotides, within 15nucleotides, within 30 nucleotides, within 50 nucleotides, within 100nucleotides). A regulatory sequence in the U6 promoter region can alsobe targeted with an gRNA. The U6-driven gRNAs may be designed in anarray format such that multiple gRNA sequences can be simultaneouslyreleased. When first delivered into target tissue/cells (left cell)gRNAs begin to accumulate while Cpf1 levels rise in the nucleus. Cpf1complexes with all of the gRNAs to mediate genome editing andself-inactivation of the CRISPR-Cpf1 plasmids.

One aspect of a self-inactivating CRISPR-Cpf1 system is expression ofsingly or in tandam array format from 1 up to 4 or more different guidesequences; e.g. up to about 20 or about 30 guides sequences. Eachindividual self inactivating guide sequence may target a differenttarget. Such may be processed from, e.g. one chimeric pol3 transcript.Pol3 promoters such as U6 or H1 promoters may be used. Pol2 promoterssuch as those mentioned throughout herein. Inverted terminal repeat(iTR) sequences may flank the Pol3 promoter-gRNA(s)-Pol2 promoter-Cpf1.

One aspect of a chimeric, tandem array transcript is that one or moreguide(s) edit the one or more target(s) while one or more selfinactivating guides inactivate the CRISPR/Cpf1 system. Thus, forexample, the described CRISPR-Cpf1 system for repairing expansiondisorders may be directly combined with the self-inactivatingCRISPR-Cpf1 system described herein. Such a system may, for example,have two guides directed to the target region for repair as well as atleast a third guide directed to self-inactivation of the CRISPR-Cpf1.Reference is made to Application Ser. No. PCT/US2014/069897, entitled“Compositions And Methods Of Use Of Crispr-Cas Systems In NucleotideRepeat Disorders,” published Dec. 12, 2014 as WO/2015/089351.

Gene Editing or Altering a Target Loci with Cpf1

The double strand break or single strand break in one of the strandsadvantageously should be sufficiently close to target position such thatcorrection occurs. In an embodiment, the distance is not more than 50,100, 200, 300, 350 or 400 nucleotides. While not wishing to be bound bytheory, it is believed that the break should be sufficiently close totarget position such that the break is within the region that is subjectto exonuclease-mediated removal during end resection. If the distancebetween the target position and a break is too great, the mutation maynot be included in the end resection and, therefore, may not becorrected, as the template nucleic acid sequence may only be used tocorrect sequence within the end resection region.

In an embodiment, in which a guide RNA and a Type V/Type VI molecule, inparticular Cpf1/C2c1/C2c2 or an ortholog or homolog thereof, preferablya Cpf1 nuclease induce a double strand break for the purpose of inducingHDR-mediated correction, the cleavage site is between 0-200 bp (e.g., 0to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200,75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the targetposition. In an embodiment, the cleavage site is between 0-100 bp (e.g.,0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50to 75 or 75 to 100 bp) away from the target position. In a furtherembodiment, two or more guide RNAs complexing with Cpf1 or an orthologor homolog thereof, may be used to induce multiplexed breaks for purposeof inducing HDR-mediated correction.

The homology arm should extend at least as far as the region in whichend resection may occur, e.g., in order to allow the resected singlestranded overhang to find a complementary region within the donortemplate. The overall length could be limited by parameters such asplasmid size or viral packaging limits. In an embodiment, a homology armmay not extend into repeated elements. Exemplary homology arm lengthsinclude a least 50, 100, 250, 500, 750 or 1000 nucleotides.

Target position, as used herein, refers to a site on a target nucleicacid or target gene (e.g., the chromosome) that is modified by a TypeV/Type VI, in particular Cpf1/C2c1/C2c2 or an ortholog or homologthereof, preferably Cpf1 molecule-dependent process. For example, thetarget position can be a modified Cpf1 molecule cleavage of the targetnucleic acid and template nucleic acid directed modification, e.g.,correction, of the target position. In an embodiment, a target positioncan be a site between two nucleotides, e.g., adjacent nucleotides, onthe target nucleic acid into which one or more nucleotides is added. Thetarget position may comprise one or more nucleotides that are altered,e.g., corrected, by a template nucleic acid. In an embodiment, thetarget position is within a target sequence (e.g., the sequence to whichthe guide RNA binds). In an embodiment, a target position is upstream ordownstream of a target sequence (e.g., the sequence to which the guideRNA binds).

A template nucleic acid, as that term is used herein, refers to anucleic acid sequence which can be used in conjunction with a TypeV/Type VI molecule, in particular Cpf1/C2c1/C2c2 or an ortholog orhomolog thereof, preferably a Cpf1 molecule and a guide RNA molecule toalter the structure of a target position. In an embodiment, the targetnucleic acid is modified to have some or all of the sequence of thetemplate nucleic acid, typically at or near cleavage site(s). In anembodiment, the template nucleic acid is single stranded. In analternate embodiment, the template nucleic acid is double stranded. Inan embodiment, the template nucleic acid is DNA, e.g., double strandedDNA. In an alternate embodiment, the template nucleic acid is singlestranded DNA.

In an embodiment, the template nucleic acid alters the structure of thetarget position by participating in homologous recombination. In anembodiment, the template nucleic acid alters the sequence of the targetposition. In an embodiment, the template nucleic acid results in theincorporation of a modified, or non-naturally occurring base into thetarget nucleic acid.

The template sequence may undergo a breakage mediated or catalyzedrecombination with the target sequence. In an embodiment, the templatenucleic acid may include sequence that corresponds to a site on thetarget sequence that is cleaved by an Cpf1 mediated cleavage event. Inan embodiment, the template nucleic acid may include sequence thatcorresponds to both, a first site on the target sequence that is cleavedin a first Cpf1 mediated event, and a second site on the target sequencethat is cleaved in a second Cpf1 mediated event.

In certain embodiments, the template nucleic acid can include sequencewhich results in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introducing a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation. Incertain embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter, enhancer, and an alteration in a cis-acting ortrans-acting control element.

A template nucleic acid having homology with a target position in atarget gene may be used to alter the structure of a target sequence. Thetemplate sequence may be used to alter an unwanted structure, e.g., anunwanted or mutant nucleotide. The template nucleic acid may includesequence which, when integrated, results in: decreasing the activity ofa positive control element; increasing the activity of a positivecontrol element; decreasing the activity of a negative control element;increasing the activity of a negative control element; decreasing theexpression of a gene; increasing the expression of a gene; increasingresistance to a disorder or disease; increasing resistance to viralentry; correcting a mutation or altering an unwanted amino acid residueconferring, increasing, abolishing or decreasing a biological propertyof a gene product, e.g., increasing the enzymatic activity of an enzyme,or increasing the ability of a gene product to interact with anothermolecule.

The template nucleic acid may include sequence which results in: achange in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or morenucleotides of the target sequence. In an embodiment, the templatenucleic acid may be 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10,70+/−10, 80+/−10, 90+/−10, 100+/−10, 110+/−10, 120+/−10, 130+/−10,140+/−10, 150+/−10, 160+/−10, 170+/−10, 180+/−10, 190+/−10, 200+/−10,210+/−10, of 220+/−10 nucleotides in length. In an embodiment, thetemplate nucleic acid may be 30+/−20, 40+/−20, 50+/−20, 60+/−20,70+/−20, 80+/−20, 90+/−20, 100+/−20, 110+/−20, 120+/−20, 130+/−20,140+/−20, I 50+/−20, 160+/−20, 170+/−20, 180+/−20, 190+/−20, 200+/−20,210+/−20, of 220+/−20 nucleotides in length. In an embodiment, thetemplate nucleic acid is 10 to 1,000, 20 to 900, 30 to 800, 40 to 700,50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, or 50 to 100nucleotides in length.

A template nucleic acid comprises the following components: [5′ homologyarm]-[replacement sequence]-[3′ homology arm]. The homology arms providefor recombination into the chromosome, thus replacing the undesiredelement, e.g., a mutation or signature, with the replacement sequence.In an embodiment, the homology arms flank the most distal cleavagesites. In an embodiment, the 3′ end of the 5′ homology arm is theposition next to the 5′ end of the replacement sequence. In anembodiment, the 5′ homology arm can extend at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000nucleotides 5′ from the 5′ end of the replacement sequence. In anembodiment, the 5′ end of the 3′ homology arm is the position next tothe 3′ end of the replacement sequence. In an embodiment, the 3′homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3′ from the 3′end of the replacement sequence.

In certain embodiments, one or both homology arms may be shortened toavoid including certain sequence repeat elements. For example, a 5′homology arm may be shortened to avoid a sequence repeat element. Inother embodiments, a 3′ homology arm may be shortened to avoid asequence repeat element. In some embodiments, both the 5′ and the 3′homology arms may be shortened to avoid including certain sequencerepeat elements.

In certain embodiments, a template nucleic acids for correcting amutation may designed for use as a single-stranded oligonucleotide. Whenusing a single-stranded oligonucleotide, 5′ and 3′ homology arms mayrange up to about 200 base pairs (bp) in length, e.g., at least 25, 50,75, 100, 125, 150, 175, or 200 bp in length.

Cpf1 Effector Protein Complex System Promoted Non-Homologous End-Joining

In certain embodiments, nuclease-induced non-homologous end-joining(NHEJ) can be used to target gene-specific knockouts. Nuclease-inducedNHEJ can also be used to remove (e.g., delete) sequence in a gene ofinterest. Generally, NHEJ repairs a double-strand break in the DNA byjoining together the two ends; however, generally, the original sequenceis restored only if two compatible ends, exactly as they were formed bythe double-strand break, are perfectly ligated. The DNA ends of thedouble-strand break are frequently the subject of enzymatic processing,resulting in the addition or removal of nucleotides, at one or bothstrands, prior to rejoining of the ends. This results in the presence ofinsertion and/or deletion (indel) mutations in the DNA sequence at thesite of the NHEJ repair. Two-thirds of these mutations typically alterthe reading frame and, therefore, produce a non-functional protein.Additionally, mutations that maintain the reading frame, but whichinsert or delete a significant amount of sequence, can destroyfunctionality of the protein. This is locus dependent as mutations incritical functional domains are likely less tolerable than mutations innon-critical regions of the protein. The indel mutations generated byNHEJ are unpredictable in nature; however, at a given break site certainindel sequences are favored and are over represented in the population,likely due to small regions of microhomology. The lengths of deletionscan vary widely; most commonly in the 1-50 bp range, but they can easilybe greater than 50 bp, e.g., they can easily reach greater than about100-200 bp. Insertions tend to be shorter and often include shortduplications of the sequence immediately surrounding the break site.However, it is possible to obtain large insertions, and in these cases,the inserted sequence has often been traced to other regions of thegenome or to plasmid DNA present in the cells.

Because NHEJ is a mutagenic process, it may also be used to delete smallsequence motifs as long as the generation of a specific final sequenceis not required. If a double-strand break is targeted near to a shorttarget sequence, the deletion mutations caused by the NHEJ repair oftenspan, and therefore remove, the unwanted nucleotides. For the deletionof larger DNA segments, introducing two double-strand breaks, one oneach side of the sequence, can result in NHEJ between the ends withremoval of the entire intervening sequence. Both of these approaches canbe used to delete specific DNA sequences; however, the error-pronenature of NHEJ may still produce indel mutations at the site of repair.

Both double strand cleaving Type V/Type VI molecule, in particularCpf1/C2c1/C2c2 or an ortholog or homolog thereof, preferably Cpf1molecules and single strand, or nickase, Type V/Type VI molecule, inparticular Cpf1/C2c1/C2c2 or an ortholog or homolog thereof, preferablyCpf1 molecules can be used in the methods and compositions describedherein to generate NHEJ-mediated indels. NHEJ-mediated indels targetedto the gene, e.g., a coding region, e.g., an early coding region of agene of interest can be used to knockout (i.e., eliminate expression of)a gene of interest. For example, early coding region of a gene ofinterest includes sequence immediately following a transcription startsite, within a first exon of the coding sequence, or within 500 bp ofthe transcription start site (e.g., less than 500, 450, 400, 350, 300,250, 200, 150, 100 or 50 bp).

In an embodiment, in which a guide RNA and Type V/Type VI molecule, inparticular Cpf1/C2c1/C2c2 or an ortholog or homolog thereof, preferablyCpf1 nuclease generate a double strand break for the purpose of inducingNHEJ-mediated indels, a guide RNA may be configured to position onedouble-strand break in close proximity to a nucleotide of the targetposition. In an embodiment, the cleavage site may be between 0-500 bpaway from the target position (e.g., less than 500, 400, 300, 200, 100,50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from thetarget position).

In an embodiment, in which two guide RNAs complexing with Type V/Type VImolecules, in particular Cpf1/C2c1/C2c2 or an ortholog or homologthereof, preferably Cpf1 nickases induce two single strand breaks forthe purpose of inducing NHEJ-mediated indels, two guide RNAs may beconfigured to position two single-strand breaks to provide for NHEJrepair a nucleotide of the target position.

Cpf1 Effector Protein Complexes can Deliver Functional Effectors

Unlike CRISPR-Cas-mediated gene knockout, which permanently eliminatesexpression by mutating the gene at the DNA level, CRISPR-Cas knockdownallows for temporary reduction of gene expression through the use ofartificial transcription factors. Mutating key residues in both DNAcleavage domains of the Cpf1 protein, such as FnCpf1 protein (e.g. theD917A and H1006A mutations of the FnCpf1 protein or D908A, E993A, D1263Aaccording to AsCpf1 protein or D832A, E925A, D947A or D1180A accordingto LbCpf1 protein) results in the generation of a catalytically inactiveCpf1. A catalytically inactive Cpf1 complexes with a guide RNA andlocalizes to the DNA sequence specified by that guide RNA's targetingdomain, however, it does not cleave the target DNA. Fusion of theinactive Cpf1 protein, such as FnCpf1 protein (e.g. the D917A and H1006Amutations) to an effector domain, e.g., a transcription repressiondomain, enables recruitment of the effector to any DNA site specified bythe guide RNA. In certain embodiments, Cpf1 may be fused to atranscriptional repression domain and recruited to the promoter regionof a gene. Especially for gene repression, it is contemplated hereinthat blocking the binding site of an endogenous transcription factorwould aid in downregulating gene expression. In another embodiment, aninactive Cpf1 can be fused to a chromatin modifying protein. Alteringchromatin status can result in decreased expression of the target gene.

In an embodiment, a guide RNA molecule can be targeted to a knowntranscription response elements (e.g., promoters, enhancers, etc.), aknown upstream activating sequences, and/or sequences of unknown orknown function that are suspected of being able to control expression ofthe target DNA.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced.

In certain embodiments, the CRISPR enzyme comprises one or moremutations selected from the group consisting of D917A, E1006A and D1225Aand/or the one or more mutations is in a RuvC domain of the CRISPRenzyme or is a mutation as otherwise as discussed herein. In someembodiments, the CRISPR enzyme has one or more mutations in a catalyticdomain, wherein when transcribed, the direct repeat sequence forms asingle stem loop and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theenzyme further comprises a functional domain. In some embodiments, thefunctional domain is a transcriptional activation domain, preferablyVP64. In some embodiments, the functional domain is a transcriptionrepression domain, preferably KRAB. In some embodiments, thetranscription repression domain is SID, or concatemers of SID (egSID4X). In some embodiments, the functional domain is an epigeneticmodifying domain, such that an epigenetic modifying enzyme is provided.In some embodiments, the functional domain is an activation domain,which may be the P65 activation domain.

Delivery of the Cpf1 Effector Protein Complex or Components Thereof

Through this disclosure and the knowledge in the art, CRISPR-Cas system,specifically the novel CRISPR systems described herein, or componentsthereof or nucleic acid molecules thereof (including, for instance HDRtemplate) or nucleic acid molecules encoding or providing componentsthereof may be delivered by a delivery system herein described bothgenerally and in detail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, forinstance a Cpf1, and/or any of the present RNAs, for instance a guideRNA, can be delivered using any suitable vector, e.g., plasmid or viralvectors, such as adeno associated virus (AAV), lentivirus, adenovirus orother viral vector types, or combinations thereof. Cpf1 and one or moreguide RNAs can be packaged into one or more vectors, e.g., plasmid orviral vectors. In some embodiments, the vector, e.g., plasmid or viralvector is delivered to the tissue of interest by, for example, anintramuscular injection, while other times the delivery is viaintravenous, transdermal, intranasal, oral, mucosal, or other deliverymethods. Such delivery may be either via a single dose, or multipledoses. One skilled in the art understands that the actual dosage to bedelivered herein may vary greatly depending upon a variety of factors,such as the vector choice, the target cell, organism, or tissue, thegeneral condition of the subject to be treated, the degree oftransformation/modification sought, the administration route, theadministration mode, the type of transformation/modification sought,etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10° particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10⁷ pu, about 4×10⁷ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding a CRISPRenzyme, operably linked to said promoter; (iii) a selectable marker;(iv) an origin of replication; and (v) a transcription terminatordownstream of and operably linked to (ii). The plasmid can also encodethe RNA components of a CRISPR complex, but one or more of these mayinstead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

The dosage used for the compositions provided herein include dosages forrepeated administration or repeat dosing. In particular embodiments, theadministration is repeated within a period of several weeks, months, oryears. Suitable assays can be performed to obtain an optimal dosageregime. Repeated administration can allow the use of lower dosage, whichcan positively affect off-target modifications.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver Cpf1 and gRNA (and, for instance, HR repairtemplate) into cells using liposomes or nanoparticles. Thus delivery ofthe CRISPR enzyme, such as a Cpf1 and/or delivery of the RNAs of theinvention may be in RNA form and via microvesicles, liposomes orparticle or particles. For example, Cpf1 mRNA and gRNA can be packagedinto liposomal particles for delivery in vivo. Liposomal transfectionreagents such as lipofectamine from Life Technologies and other reagentson the market can effectively deliver RNA molecules into the liver.

Means of delivery of RNA also preferred include delivery of RNA viaparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y.,Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles forsmall interfering RNA delivery to endothelial cells, Advanced FunctionalMaterials, 19: 3112-3118, 2010) or exosomes (Schroeder, A., Levins, C.,Cortez, C., Langer, R., and Anderson, D., Lipid-based nanotherapeuticsfor siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID:20059641). Indeed, exosomes have been shown to be particularly useful indelivery siRNA, a system with some parallels to the CRISPR system. Forinstance, El-Andaloussi S, et al. (“Exosome-mediated delivery of siRNAin vitro and in vivo.” Nat Protoc. 2012 December; 7(12):2112-26. doi:10.1038/nprot.2012.131. Epub 2012 Nov. 15) describe how exosomes arepromising tools for drug delivery across different biological barriersand can be harnessed for delivery of siRNA in vitro and in vivo. Theirapproach is to generate targeted exosomes through transfection of anexpression vector, comprising an exosomal protein fused with a peptideligand. The exosomes are then purify and characterized from transfectedcell supernatant, then RNA is loaded into the exosomes. Delivery oradministration according to the invention can be performed withexosomes, in particular but not limited to the brain. Vitamin E(α-tocopherol) may be conjugated with CRISPR Cas and delivered to thebrain along with high density lipoprotein (HDL), for example in asimilar manner as was done by Uno et al. (HUMAN GENE THERAPY 22:711-719(June 2011)) FOR DELIVERING SHORT-INTERFERING RNA (SIRNA) TO THE BRAIN.MICE WERE INFUSED VIA OSMOTIC MINIPUMPS (MODEL 1007D; ALZET, CUPERTINO,CALIF.) FILLED with phosphate-buffered saline (PBS) or free TocsiBACE orToc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet). Abrain-infusion cannula was placed about 0.5 mm posterior to the bregmaat midline for infusion into the dorsal third ventricle. Uno et al.found that as little as 3 nmol of Toc-siRNA with HDL could induce atarget reduction in comparable degree by the same ICV infusion method. Asimilar dosage of CRISPR Cas conjugated to α-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of CRISPR Cas targeted to the brain may be contemplated. Zou et al.((HUMAN GENE THERAPY 22:465-475 (April 2011)) describes a method oflentiviral-mediated delivery of short-hairpin RNAs targeting PKCγ for invivo gene silencing in the spinal cord of rats. Zou et al. administeredabout 10 μl of a recombinant lentivirus having a titer of 1×10⁹transducing units (TU)/ml by an intrathecal catheter. A similar dosageof CRISPR Cas expressed in a lentiviral vector targeted to the brain maybe contemplated for humans in the present invention, for example, about10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having atiter of 1×10⁹ transducing units (TU)/ml may be contemplated.

Preassembled recombinant CRISPR-Cpf1 complexes comprising Cpf1 and crRNAmay be transfected, for example by electroporation, resulting in highmutation rates and absence of detectable off-target mutations. Hur, J.K. et al, Targeted mutagenesis in mice by electroporation of Cpf1ribonucleoproteins, Nat Biotechnol. 2016 Jun. 6. doi: 10.1038/nbt.3596.[Epub ahead of print]

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g. byinjection. Injection can be performed stereotactically via a craniotomy.

Enhancing NHEJ or HR efficiency is also helpful for delivery. It ispreferred that NHEJ efficiency is enhanced by co-expressingend-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011August; 188(4): 787-797). It is preferred that HR efficiency isincreased by transiently inhibiting NHEJ machineries such as Ku70 andKu86. HR efficiency can also be increased by co-expressing prokaryoticor eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Packaging and Promoters

Ways to package inventive Cpf1 coding nucleic acid molecules, e.g., DNA,into vectors, e.g., viral vectors, to mediate genome modification invivo include:

-   -   To achieve NHEJ-mediated gene knockout:    -   Single virus vector:    -   Vector containing two or more expression cassettes:    -   Promoter-Cpf1 coding nucleic acid molecule-terminator    -   Promoter-gRNA 1-terminator    -   Promoter-gRNA2-terminator    -   Promoter-gRNA(N)-terminator (up to size limit of vector)    -   Double virus vector:    -   Vector 1 containing one expression cassette for driving the        expression of Cpf1    -   Promoter-Cpf1 coding nucleic acid molecule-terminator    -   Vector 2 containing one more expression cassettes for driving        the expression of one or more guideRNAs    -   Promoter-gRNA 1-terminator    -   Promoter-gRNA(N)-terminator (up to size limit of vector)    -   To mediate homology-directed repair.    -   In addition to the single and double virus vector approaches        described above, an additional vector can be used to deliver a        homology-direct repair template.

The promoter used to drive Cpf1 coding nucleic acid molecule expressioncan include:

AAV ITR can serve as a promoter: this is advantageous for eliminatingthe need for an additional promoter element (which can take up space inthe vector). The additional space freed up can be used to drive theexpression of additional elements (gRNA, etc.). Also, ITR activity isrelatively weaker, so can be used to reduce potential toxicity due toover expression of Cpf1.

For ubiquitous expression, promoters that can be used include: CMV, CAG,CBh, PGK, SV40, Ferritin heavy or light chains, etc.

For brain or other CNS expression, can use promoters: SynapsinI for allneurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT forGABAergic neurons, etc.

For liver expression, can use Albumin promoter.

For lung expression, can use SP-B.

For endothelial cells, can use ICAM.

For hematopoietic cells can use IFNbeta or CD45.

For Osteoblasts can one can use the OG-2.

The promoter used to drive guide RNA can include:

Pol III promoters such as U6 or H1

Use of Pol II promoter and intronic cassettes to express gRNA

Adeno Associated Virus (AAV)

Cpf1 and one or more guide RNA can be delivered using adeno associatedvirus (AAV), lentivirus, adenovirus or other plasmid or viral vectortypes, in particular, using formulations and doses from, for example,U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat.No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946(formulations, doses for DNA plasmids) and from clinical trials andpublications regarding the clinical trials involving lentivirus, AAV andadenovirus. For examples, for AAV, the route of administration,formulation and dose can be as in U.S. Pat. No. 8,454,972 and as inclinical trials involving AAV. For Adenovirus, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g. a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of Cpf1 can be driven by a cell-typespecific promoter. For example, liver-specific expression might use theAlbumin promoter and neuron-specific expression (e.g. for targeting CNSdisorders) might use the Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response) and    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cpf1 aswell as a promoter and transcription terminator have to be all fit intothe same viral vector. Constructs larger than 4.5 or 4.75 Kb will leadto significantly reduced virus production. SpCas9 is quite large, thegene itself is over 4.1 Kb, which makes it difficult for packing intoAAV. Therefore embodiments of the invention include utilizing homologsof Cpf1 that are shorter.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4 C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinfectious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the CRISPR-Cas system of the presentinvention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the CRISPR-Cas system of the presentinvention. A minimum of 2.5×106 CD34+ cells per kilogram patient weightmay be collected and prestimulated for 16 to 20 hours in X-VIVO 15medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×106 cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm2 tissue culture flasks coated with fibronectin (25mg/cm2) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The CRISPR enzyme, for instance a Cpf1, and/or any of thepresent RNAs, for instance a guide RNA, can also be delivered in theform of RNA. Cpf1 mRNA can be generated using in vitro transcription.For example, Cpf1 mRNA can be synthesized using a PCR cassettecontaining the following elements: T7_promoter-kozak sequence(GCCACC)-Cpf1-3′ UTR from beta globin-polyA tail (a string of 120 ormore adenines). The cassette can be used for transcription by T7polymerase. Guide RNAs can also be transcribed using in vitrotranscription from a cassette containing T7_promoter-GG-guide RNAsequence.

To enhance expression and reduce possible toxicity, the CRISPRenzyme-coding sequence and/or the guide RNA can be modified to includeone or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlmanand Carmen Barnes et al. Nature Nanotechnology (2014) published online11 May 2014, doi:10.1038/nnano.2014.84, concerning particles, methods ofmaking and using them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Particles

It will be appreciated that reference made herein to particles ornanoparticles can be interchangeable, where appropriate. CRISPR enzymemRNA and guide RNA may be delivered simultaneously using particles orlipid envelopes; for instance, CRISPR enzyme and RNA of the invention,e.g., as a complex, can be delivered via a particle as in Dahlman etal., WO2015089419 A2 and documents cited therein, such as 7C1 (see,e.g., James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology(2014) published online 11 May 2014, doi:10.1038/nnano.2014.84), e.g.,delivery particle comprising lipid or lipidoid and hydrophilic polymer,e.g., cationic lipid and hydrophilic polymer, for instance wherein thecationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP) or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/orwherein the hydrophilic polymer comprises ethylene glycol orpolyethylene glycol (PEG); and/or wherein the particle further comprisescholesterol (e.g., particle from formulation 1=DOTAP 100, DMPC 0, PEG 0,Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10,Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol5), wherein particles are formed using an efficient, multistep processwherein first, effector protein and RNA are mixed together, e.g., at a1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g.,in sterile, nuclease free 1×PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes).

Nucleic acid-targeting effector proteins (such as a Type V protein suchCpf1) mRNA and guide RNA may be delivered simultaneously using particlesor lipid envelopes. Examples of suitable particles include but are notlimited to those described in U.S. Pat. No. 9,301,923.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured nanoparticles with a poly(β-amino ester) (PBAE) coreenveloped by a phospholipid bilayer shell. These were developed for invivo mRNA delivery. The pH-responsive PBAE component was chosen topromote endosome disruption, while the lipid surface layer was selectedto minimize toxicity of the polycation core. Such are, therefore,preferred for delivering RNA of the present invention.

In one embodiment, particles/nanoparticles based on self assemblingbioadhesive polymers are contemplated, which may be applied to oraldelivery of peptides, intravenous delivery of peptides and nasaldelivery of peptides, all to the brain. Other embodiments, such as oralabsorption and ocular delivery of hydrophobic drugs are alsocontemplated. The molecular envelope technology involves an engineeredpolymer envelope which is protected and delivered to the site of thedisease (see, e.g., Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026;Siew, A., et al. Mol Pharm, 2012. 9(1):14-28; Lalatsa, A., et al. JContr Rel, 2012. 161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012.9(6):1665-80; Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74;Garrett, N. L., et al. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N.L., et al. J Raman Spect, 2012. 43(5):681-688; Ahmad, S., et al. J RoyalSoc Interface 2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv,2006. 3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9and Uchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses ofabout 5 mg/kg are contemplated, with single or multiple doses, dependingon the target tissue.

In one embodiment, particles/nanoparticles that can deliver RNA to acancer cell to stop tumor growth developed by Dan Anderson's lab at MITmay be used/and or adapted to the CRISPR Cas system of the presentinvention. In particular, the Anderson lab developed fully automated,combinatorial systems for the synthesis, purification, characterization,and formulation of new biomaterials and nanoformulations. See, e.g.,Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6;Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., NanoLett. 2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the CRISPR Cas system of the present invention. Inone aspect, the aminoalcohol lipidoid compounds are combined with anagent to be delivered to a cell or a subject to form microparticles,nanoparticles, liposomes, or micelles. The agent to be delivered by theparticles, liposomes, or micelles may be in the form of a gas, liquid,or solid, and the agent may be a polynucleotide, protein, peptide, orsmall molecule. The minoalcohol lipidoid compounds may be combined withother aminoalcohol lipidoid compounds, polymers (synthetic or natural),surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to formthe particles. These particles may then optionally be combined with apharmaceutical excipient to form a pharmaceutical composition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the CRISPR Cassystem of the present invention. In some embodiments, sugar-basedparticles may be used, for example GalNAc, as described herein and withreference to WO2014118272 (incorporated herein by reference) and Nair, JK et al., 2014, Journal of the American Chemical Society 136 (49),16958-16961) and the teaching herein, especially in respect of deliveryapplies to all particles unless otherwise apparent.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe CRISPR Cas system of the present invention. Doses of about 0.01 toabout 1 mg per kg of body weight administered intravenously arecontemplated. Medications to reduce the risk of infusion-relatedreactions are contemplated, such as dexamethasone, acetampinophen,diphenhydramine or cetirizine, and ranitidine are contemplated. Multipledoses of about 0.3 mg per kilogram every 4 weeks for five doses are alsocontemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding CRISPR Cas to the liver. A dosage of about four doses of 6mg/kg of the LNP every two weeks may be contemplated. Tabernero et al.demonstrated that tumor regression was observed after the first 2 cyclesof LNPs dosed at 0.7 mg/kg, and by the end of 6 cycles the patient hadachieved a partial response with complete regression of the lymph nodemetastasis and substantial shrinkage of the liver tumors. A completeresponse was obtained after 40 doses in this patient, who has remainedin remission and completed treatment after receiving doses over 26months. Two patients with RCC and extrahepatic sites of diseaseincluding kidney, lung, and lymph nodes that were progressing followingprior therapy with VEGF pathway inhibitors had stable disease at allsites for approximately 8 to 12 months, and a patient with PNET andliver metastases continued on the extension study for 18 months (36doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), anddimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). It has been shownthat LNP siRNA systems containing these lipids exhibit remarkablydifferent gene silencing properties in hepatocytes in vivo, withpotencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR Cas encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificCRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA,DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG orPEG-C-DOMG at 40:10:40:10 molar ratios). When required, 0.2% SP-DiOC18(Invitrogen, Burlington, Canada) may be incorporated to assess cellularuptake, intracellular delivery, and biodistribution. Encapsulation maybe performed by dissolving lipid mixtures comprised of cationiclipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanolto a final lipid concentration of 10 mmol/l. This ethanol solution oflipid may be added drop-wise to 50 mmol/l citrate, pH 4.0 to formmultilamellar vesicles to produce a final concentration of 30% ethanolvol/vol. Large unilamellar vesicles may be formed following extrusion ofmultilamellar vesicles through two stacked 80 nm Nuclepore polycarbonatefilters using the Extruder (Northern Lipids, Vancouver, Canada).Encapsulation may be achieved by adding RNA dissolved at 2 mg/ml in 50mmol/l citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise toextruded preformed large unilamellar vesicles and incubation at 31° C.for 30 minutes with constant mixing to a final RNA/lipid weight ratio of0.06/1 wt/wt. Removal of ethanol and neutralization of formulationbuffer were performed by dialysis against phosphate-buffered saline(PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulosedialysis membranes. Nanoparticle size distribution may be determined bydynamic light scattering using a NICOMP 370 particle sizer, thevesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing,Santa Barbara, Calif.). The particle size for all three LNP systems maybe ˜70 nm in diameter. RNA encapsulation efficiency may be determined byremoval of free RNA using VivaPureD MiniH columns (Sartorius StedimBiotech) from samples collected before and after dialysis. Theencapsulated RNA may be extracted from the eluted nanoparticles andquantified at 260 nm. RNA to lipid ratio was determined by measurementof cholesterol content in vesicles using the Cholesterol E enzymaticassay from Wako Chemicals USA (Richmond, Va.). In conjunction with theherein discussion of LNPs and PEG lipids, PEGylated liposomes or LNPsare likewise suitable for delivery of a CRISPR-Cas system or componentsthereof.

Preparation of large LNPs may be used/and or adapted from Rosin et al,Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. Alipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at an RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other nanoparticles(particularly gold nanoparticles) are also contemplated as a means todelivery CRISPR-Cas system to intended targets. Significant data showthat AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs,based upon nucleic acid-functionalized gold nanoparticles, are useful.Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling nanoparticles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of CRISPR Cas is envisioned for deliveryin the self-assembling nanoparticles of Schiffelers et al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA nanoparticles maybe formed by using cyclodextrin-containing polycations. Typically,nanoparticles were formed in water at a charge ratio of 3 (+/−) and ansiRNA concentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted nanoparticles were modifiedwith Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5%(wt/vol) glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted nanoparticle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetednanoparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The nanoparticles consist of a synthetic deliverysystem containing: (1) a linear, cyclodextrin-based polymer (CDP), (2) ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the nanoparticle to engage TF receptors (TFR) on the surfaceof the cancer cells, (3) a hydrophilic polymer (polyethylene glycol(PEG) used to promote nanoparticle stability in biological fluids), and(4) siRNA designed to reduce the expression of the RRM2 (sequence usedin the clinic was previously denoted siR2B+5). The TFR has long beenknown to be upregulated in malignant cells, and RRM2 is an establishedanti-cancer target. These nanoparticles (clinical version denoted asCALAA-01) have been shown to be well tolerated in multi-dosing studiesin non-human primates. Although a single patient with chronic myeloidleukaemia has been administered siRNAby liposomal delivery, Davis etal.'s clinical trial is the initial human trial to systemically deliversiRNA with a targeted delivery system and to treat patients with solidcancer. To ascertain whether the targeted delivery system can provideeffective delivery of functional siRNA to human tumours, Davis et al.investigated biopsies from three patients from three different dosingcohorts; patients A, B and C, all of whom had metastatic melanoma andreceived CALAA-01 doses of 18, 24 and 30 mg m⁻² siRNA, respectively.Similar doses may also be contemplated for the CRISPR Cas system of thepresent invention. The delivery of the invention may be achieved withnanoparticles containing a linear, cyclodextrin-based polymer (CDP), ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the nanoparticle to engage TF receptors (TFR) on the surfaceof the cancer cells and/or a hydrophilic polymer (for example,polyethylene glycol (PEG) used to promote nanoparticle stability inbiological fluids).

In terms of this invention, it is preferred to have one or morecomponents of CRISPR complex, e.g., CRISPR enzyme or mRNA or guide RNAdelivered using nanoparticles or lipid envelopes. Other delivery systemsor vectors are may be used in conjunction with the nanoparticle aspectsof the invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, nanoparticles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm.

Nanoparticles encompassed in the present invention may be provided indifferent forms, e.g., as solid nanoparticles (e.g., metal such assilver, gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of nanoparticles, or combinations thereof. Metal,dielectric, and semiconductor nanoparticles may be prepared, as well ashybrid structures (e.g., core-shell nanoparticles). Nanoparticles madeof semiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft nanoparticles have been manufactured, and are withinthe scope of the present invention. A prototype nanoparticle ofsemi-solid nature is the liposome. Various types of liposomenanoparticles are currently used clinically as delivery systems foranticancer drugs and vaccines. Nanoparticles with one half hydrophilicand the other half hydrophobic are termed Janus particles and areparticularly effective for stabilizing emulsions. They can self-assembleat water/oil interfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid.

U.S. Pat. No. 6,007,845, incorporated herein by reference, providesparticles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material.

U.S. Pat. No. 5,855,913, incorporated herein by reference, provides aparticulate composition having aerodynamically light particles having atap density of less than 0.4 g/cm3 with a mean diameter of between 5 μmand 30 μm, incorporating a surfactant on the surface thereof for drugdelivery to the pulmonary system.

U.S. Pat. No. 5,985,309, incorporated herein by reference, providesparticles incorporating a surfactant and/or a hydrophilic or hydrophobiccomplex of a positively or negatively charged therapeutic or diagnosticagent and a charged molecule of opposite charge for delivery to thepulmonary system.

U.S. Pat. No. 5,543,158, incorporated herein by reference, providesbiodegradable injectable particles having a biodegradable solid corecontaining a biologically active material and poly(alkylene glycol)moieties on the surface.

WO2012135025 (also published as US20120251560), incorporated herein byreference, describes conjugated polyethyleneimine (PEI) polymers andconjugated aza-macrocycles (collectively referred to as “conjugatedlipomer” or “lipomers”). In certain embodiments, it can envisioned thatsuch conjugated lipomers can be used in the context of the CRISPR-Cassystem to achieve in vitro, ex vivo and in vivo genomic perturbations tomodify gene expression, including modulation of protein expression.

In one embodiment, the nanoparticle may be epoxide-modifiedlipid-polymer, advantageously 7C1 (see, e.g., James E. Dahlman andCarmen Barnes et al. Nature Nanotechnology (2014) published online 11May 2014, doi:10.1038/nnano.2014.84). C71 was synthesized by reactingC15 epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce nanoparticles (diameter between 35and 60 nm) that were stable in PBS solution for at least 40 days.

An epoxide-modified lipid-polymer may be utilized to deliver theCRISPR-Cas system of the present invention to pulmonary, cardiovascularor renal cells, however, one of skill in the art may adapt the system todeliver to other target organs. Dosage ranging from about 0.05 to about0.6 mg/kg are envisioned. Dosages over several days or weeks are alsoenvisioned, with a total dosage of about 2 mg/kg.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (WIC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by nanoparticle tracking analysis (NTA)and electron microscopy. Alvarez-Erviti et al. obtained 6-12 μg ofexosomes (measured based on protein concentration) per 10⁶ cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 μF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the CRISPR-Cas system of the present invention to therapeutictargets, especially neurodegenerative diseases. A dosage of about 100 to1000 mg of CRISPR Cas encapsulated in about 100 to 1000 mg of RVGexosomes may be contemplated for the present invention.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention.

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of CRISPR Cas into exosomes may beconducted similarly to siRNA. The exosomes may be co-cultured withmonocytes and lymphocytes isolated from the peripheral blood of healthydonors. Therefore, it may be contemplated that exosomes containingCRISPR Cas may be introduced to monocytes and lymphocytes of andautologously reintroduced into a human. Accordingly, delivery oradministration according to the invention may be performed using plasmaexosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long.These particles allow delivery of a transgene to the entire brain afteran intravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR family of nucleases to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the CRISPR Cas system or components thereof maybe administered in liposomes, such as a stable nucleic-acid-lipidparticle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology,Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3or 5 mg/kg/day of a specific CRISPR Cas targeted in a SNALP arecontemplated. The daily treatment may be over about three days and thenweekly for about five weeks. In another embodiment, a specific CRISPRCas encapsulated SNALP) administered by intravenous injection to atdoses of about 1 or 2.5 mg/kg are also contemplated (see, e.g.,Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALPformulation may contain the lipids 3-N-[(wmethoxypoly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar per cent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine(Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, andcationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g.,Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kgtotal CRISPR Cas per dose administered as, for example, a bolusintravenous infusion may be contemplated.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC;Avanti Polar Lipids Inc.), PEG-cDMA, and1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g.,Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for invivo studies may comprise a final lipid/RNA mass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC)—both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≥0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the CRISPR Cas system of the presentinvention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate CRISPR Cas or components thereof or nucleicacid molecule(s) coding therefor e.g., similar to SiRNA (see, e.g.,Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and hence may beemployed in the practice of the invention. A preformed vesicle with thefollowing lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11±0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume: 29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the CRISPR Cassystem of the present invention or component(s) thereof or nucleic acidmolecule(s) coding therefor to form lipid nanoparticles (LNPs). Lipidsinclude, but are not limited to, DLin-KC2-DMA4, C12-200 and colipidsdisteroylphosphatidyl choline, cholesterol, and PEG-DMG may beformulated with CRISPR Cas instead of siRNA (see, e.g., Novobrantseva,Molecular Therapy—Nucleic Acids (2012) 1, e4; doi:10.1038/mtna.2011.3)using a spontaneous vesicle formation procedure. The component molarratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG). The final lipid:siRNAweight ratio may be ˜12:1 and 9:1 in the case of DLin-KC2-DMA andC12-200 lipid nanoparticles (LNPs), respectively. The formulations mayhave mean particle diameters of ˜80 nm with >90% entrapment efficiency.A 3 mg/kg dose may be contemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The CRISPR Cas system or components thereof or nucleic acid molecule(s)coding therefor may be delivered encapsulated in PLGA Microspheres suchas that further described in US published applications 20130252281 and20130245107 and 20130244279 (assigned to Moderna Therapeutics) whichrelate to aspects of formulation of compositions comprising modifiednucleic acid molecules which may encode a protein, a protein precursor,or a partially or fully processed form of the protein or a proteinprecursor. The formulation may have a molar ratio 50:10:38.5:1.5-3.0(cationic lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipidmay be selected from, but is not limited to PEG-c-DOMG, PEG-DMG. Thefusogenic lipid may be DSPC. See also, Schrum et al., Delivery andFormulation of Engineered Nucleic Acids, US published application20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesised from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of CRISPR Cas system(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of CRISPR Cas system(s) or component(s)thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116) (However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines):

-   -   (1) One day before treatment, plate 1×10⁵ cells per well in a        48-well plate.    -   (2) On the day of treatment, dilute purified +36 GFP protein in        serumfree media to a final concentration 200 nM. Add RNA to a        final concentration of 50 nM. Vortex to mix and incubate at room        temperature for 10 min.    -   (3) During incubation, aspirate media from cells and wash once        with PBS.    -   (4) Following incubation of +36 GFP and RNA, add the protein-RNA        complexes to cells.    -   (5) Incubate cells with complexes at 37° C. for 4 h.    -   (6) Following incubation, aspirate the media and wash three        times with 20 U/mL heparin PBS. Incubate cells with        serum-containing media for a further 48 h or longer depending        upon the assay for activity.    -   (7) Analyze cells by immunoblot, qPCR, phenotypic assay, or        other appropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications:

-   -   (1) One day before treatment, plate 1×10⁵ per well in a 48-well        plate.    -   (2) On the day of treatment, dilute purified        36 GFP protein in serumfree media to a final concentration 2 mM.        Add 1 mg of plasmid DNA. Vortex to mix and incubate at room        temperature for 10 min.    -   (3) During incubation, aspirate media from cells and wash once        with PBS.    -   (4) Following incubation of        36 GFP and plasmid DNA, gently add the protein-DNA complexes to        cells.    -   (5) Incubate cells with complexes at 37 C for 4 h.    -   (6) Following incubation, aspirate the media and wash with PBS.        Incubate cells in serum-containing media and incubate for a        further 24-48 h.    -   (7) Analyze plasmid delivery (e.g., by plasmid-driven gene        expression) as appropriate.

See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106,6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe CRISPR Cas system of the present invention. These systems of Dr. Luiand documents herein in conjunction with herein teaching can be employedin the delivery of CRISPR Cas system(s) or component(s) thereof ornucleic acid molecule(s) coding therefor.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins,particles, including nanoparticles, liposomes, chromophores, smallmolecules and radioactive materials. In aspects of the invention, thecargo may also comprise any component of the CRISPR Cas system or theentire functional CRISPR Cas system. Aspects of the present inventionfurther provide methods for delivering a desired cargo into a subjectcomprising: (a) preparing a complex comprising the cell penetratingpeptide of the present invention and a desired cargo, and (b) orally,intraarticularly, intraperitoneally, intrathecally, intrarterially,intranasally, intraparenchymally, subcutaneously, intramuscularly,intravenously, dermally, intrarectally, or topically administering thecomplex to a subject. The cargo is associated with the peptides eitherthrough chemical linkage via covalent bonds or through non-covalentinteractions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP, MMcontrast agents, or quantum dots. CPPs hold great potential as in vitroand in vivo delivery vectors for use in research and medicine. CPPstypically have an amino acid composition that either contains a highrelative abundance of positively charged amino acids such as lysine orarginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only apolar residues, with low net charge or have hydrophobicamino acid groups that are crucial for cellular uptake. One of theinitial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPPscan be used to deliver the CRISPR-Cas system or components thereof. ThatCPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged particles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the CRISPR Cas system or component(s) thereof or nucleicacid molecule(s) coding therefor. For example, US Patent Publication20110195123 discloses an implantable medical device which elutes a druglocally and in prolonged period is provided, including several types ofsuch a device, the treatment modes of implementation and methods ofimplantation. The device comprising of polymeric substrate, such as amatrix for example, that is used as the device body, and drugs, and insome cases additional scaffolding materials, such as metals oradditional polymers, and materials to enhance visibility and imaging. Animplantable delivery device can be advantageous in providing releaselocally and over a prolonged period, where drug is released directly tothe extracellular matrix (ECM) of the diseased area such as tumor,inflammation, degeneration or for symptomatic objectives, or to injuredsmooth muscle cells, or for prevention. One kind of drug is RNA, asdisclosed above, and this system may be used/and or adapted to theCRISPR Cas system of the present invention. The modes of implantation insome embodiments are existing implantation procedures that are developedand used today for other treatments, including brachytherapy and needlebiopsy. In such cases the dimensions of the new implant described inthis invention are similar to the original implant. Typically a fewdevices are implanted during the same treatment procedure.

US Patent Publication 20110195123, provides a drug delivery implantableor insertable system, including systems applicable to a cavity such asthe abdominal cavity and/or any other type of administration in whichthe drug delivery system is not anchored or attached, comprising abiostable and/or degradable and/or bioabsorbable polymeric substrate,which may for example optionally be a matrix. It should be noted thatthe term “insertion” also includes implantation. The drug deliverysystem is preferably implemented as a “Loder” as described in US PatentPublication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of US Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group comprising,consisting essentially of, or consisting of (as non-limiting examplesonly, as optionally any site within the body may be suitable forimplanting a Loder): 1. brain at degenerative sites like in Parkinson orAlzheimer disease at the basal ganglia, white and gray matter; 2. spineas in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervixto prevent HPV infection; 4. active and chronic inflammatory joints; 5.dermis as in the case of psoriasis; 6. sympathetic and sensoric nervoussites for analgesic effect; 7. Intra osseous implantation; 8. acute andchronic infection sites; 9. Intra vaginal; 10. Inner ear-auditorysystem, labyrinth of the inner ear, vestibular system; 11. Intratracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder;14. biliary system; 15. parenchymal tissue including and not limited tothe kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18.dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21.Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominalcavity (for example but without limitation, for ovary cancer); 24. Intraesophageal and 25. Intra rectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of US Patent Publication 20110195123, thedrug preferably comprises a RNA, for example for localized cancer casesin breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the CRISPR Cas system of the presentinvention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the CRISPR Cas system of the present invention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the CRISPR Cas system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used/and or adapted to the CRISPR Cas system of the presentinvention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

Patient-Specific Screening Methods

A nucleic acid-targeting system that targets DNA, e.g., trinucleotiderepeats can be used to screen patients or patent samples for thepresence of such repeats. The repeats can be the target of the RNA ofthe nucleic acid-targeting system, and if there is binding thereto bythe nucleic acid-targeting system, that binding can be detected, tothereby indicate that such a repeat is present. Thus, a nucleicacid-targeting system can be used to screen patients or patient samplesfor the presence of the repeat. The patient can then be administeredsuitable compound(s) to address the condition; or, can be administered anucleic acid-targeting system to bind to and cause insertion, deletionor mutation and alleviate the condition.

The invention uses nucleic acids to bind target DNA sequences.

CRISPR Effector Protein mRNA and Guide RNA

CRISPR enzyme mRNA and guide RNA might also be delivered separately.CRISPR enzyme mRNA can be delivered prior to the guide RNA to give timefor CRISPR enzyme to be expressed. CRISPR enzyme mRNA might beadministered 1-12 hours (preferably around 2-6 hours) prior to theadministration of guide RNA.

Alternatively, CRISPR enzyme mRNA and guide RNA can be administeredtogether. Advantageously, a second booster dose of guide RNA can beadministered 1-12 hours (preferably around 2-6 hours) after the initialadministration of CRISPR enzyme mRNA+guide RNA.

The CRISPR effector protein of the present invention, i.e. Cpf1 effectorprotein is sometimes referred to herein as a CRISPR Enzyme. It will beappreciated that the effector protein is based on or derived from anenzyme, so the term ‘effector protein’ certainly includes ‘enzyme’ insome embodiments. However, it will also be appreciated that the effectorprotein may, as required in some embodiments, have DNA or RNA binding,but not necessarily cutting or nicking, activity, including a dead-Caseffector protein function.

Additional administrations of CRISPR enzyme mRNA and/or guide RNA mightbe useful to achieve the most efficient levels of genome modification.In some embodiments, phenotypic alteration is preferably the result ofgenome modification when a genetic disease is targeted, especially inmethods of therapy and preferably where a repair template is provided tocorrect or alter the phenotype.

In some embodiments diseases that may be targeted include thoseconcerned with disease-causing splice defects.

In some embodiments, cellular targets include HemopoieticStem/Progenitor Cells (CD34+); Human T cells; and Eye (retinalcells)—for example photoreceptor precursor cells.

In some embodiments Gene targets include: Human Beta Globin—BBB (fortreating Sickle Cell Anemia, including by stimulating gene-conversion(using closely related HBD gene as an endogenous template)); CD3(T-Cells); and CEP920—retina (eye).

In some embodiments disease targets also include: cancer; Sickle CellAnemia (based on a point mutation); HIV; Beta-Thalassemia; andophthalmic or ocular disease—for example Leber Congenital Amaurosis(LCA)-causing Splice Defect.

In some embodiments delivery methods include: Cationic Lipid Mediated“direct” delivery of Enzyme-Guide complex (RiboNucleoProtein) andelectroporation of plasmid DNA.

Inventive methods can further comprise delivery of templates, such asrepair templates, which may be dsODN or ssODN, see below. Delivery oftemplates may be via the cotemporaneous or separate from delivery of anyor all the CRISPR enzyme or guide and via the same delivery mechanism ordifferent. In some embodiments, it is preferred that the template isdelivered together with the guide, and, preferably, also the CRISPRenzyme. An example may be an AAV vector.

Inventive methods can further comprise: (a) delivering to the cell adouble-stranded oligodeoxynucleotide (dsODN) comprising overhangscomplimentary to the overhangs created by said double strand break,wherein said dsODN is integrated into the locus of interest; or —(b)delivering to the cell a single-stranded oligodeoxynucleotide (ssODN),wherein said ssODN acts as a template for homology directed repair ofsaid double strand break. Inventive methods can be for the prevention ortreatment of disease in an individual, optionally wherein said diseaseis caused by a defect in said locus of interest. Inventive methods canbe conducted in vivo in the individual or ex vivo on a cell taken fromthe individual, optionally wherein said cell is returned to theindividual.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of CRISPR enzyme mRNA and guide RNAdelivered. Optimal concentrations of CRISPR enzyme mRNA and guide RNAcan be determined by testing different concentrations in a cellular oranimal model and using deep sequencing the analyze the extent ofmodification at potential off-target genomic loci. For example, for theguide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′ (SEQ ID NO: 23) inthe EMX1 gene of the human genome, deep sequencing can be used to assessthe level of modification at the following two off-target loci, 1:5′-GAGTCCTAGCAGGAGAAGAA-3′ (SEQ ID NO: 24) and 2:5′-GAGTCTAAGCAGAAGAAGAA-3′ (SEQ ID NO: 25). The concentration that givesthe highest level of on-target modification while minimizing the levelof off-target modification should be chosen for in vivo delivery.

Inducible Systems

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283, and WO 2014/018423 A2which is hereby incorporated by reference in its entirety.

Self-Inactivating Systems

Once all copies of a gene in the genome of a cell have been edited,continued CRISRP/Cpf1p expression in that cell is no longer necessary.Indeed, sustained expression would be undesirable in case of off-targeteffects at unintended genomic sites, etc. Thus time-limited expressionwould be useful. Inducible expression offers one approach, but inaddition Applicants have engineered a Self-Inactivating CRISPR systemthat relies on the use of a non-coding guide target sequence within theCRISPR vector itself. Thus, after expression begins, the CRISPR-Cassystem will lead to its own destruction, but before destruction iscomplete it will have time to edit the genomic copies of the target gene(which, with a normal point mutation in a diploid cell, requires at mosttwo edits). Simply, the self inactivating CRISPR-Cas system includesadditional RNA (i.e., guide RNA) that targets the coding sequence forthe CRISPR enzyme itself or that targets one or more non-coding guidetarget sequences complementary to unique sequences present in one ormore of the following:

(a) within the promoter driving expression of the non-coding RNAelements,(b) within the promoter driving expression of the Cpf1 effector proteingene,(c) within 100 bp of the ATG translational start codon in the Cpf1effector protein coding sequence,(d) within the inverted terminal repeat (iTR) of a viral deliveryvector, e.g., in the AAV genome.

Furthermore, that RNA can be delivered via a vector, e.g., a separatevector or the same vector that is encoding the CRISPR complex. Whenprovided by a separate vector, the CRISPR RNA that targets Casexpression can be administered sequentially or simultaneously. Whenadministered sequentially, the CRISPR RNA that targets Cas expression isto be delivered after the CRISPR RNA that is intended for e.g. geneediting or gene engineering. This period may be a period of minutes(e.g. 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60minutes). This period may be a period of hours (e.g. 2 hours, 4 hours, 6hours, 8 hours, 12 hours, 24 hours). This period may be a period of days(e.g. 2 days, 3 days, 4 days, 7 days). This period may be a period ofweeks (e.g. 2 weeks, 3 weeks, 4 weeks). This period may be a period ofmonths (e.g. 2 months, 4 months, 8 months, 12 months). This period maybe a period of years (2 years, 3 years, 4 years). In this fashion, theCas enzyme associates with a first gRNA capable of hybridizing to afirst target, such as a genomic locus or loci of interest and undertakesthe function(s) desired of the CRISPR-Cas system (e.g., geneengineering); and subsequently the Cas enzyme may then associate withthe second gRNA capable of hybridizing to the sequence comprising atleast part of the Cas or CRISPR cassette. Where the guide RNA targetsthe sequences encoding expression of the Cas protein, the enzyme becomesimpeded and the system becomes self inactivating. In the same manner,CRISPR RNA that targets Cas expression applied via, for exampleliposome, lipofection, particles, microvesicles as explained herein, maybe administered sequentially or simultaneously. Similarly,self-inactivation may be used for inactivation of one or more guide RNAused to target one or more targets.

In some aspects, a single gRNA is provided that is capable ofhybridization to a sequence downstream of a CRISPR enzyme start codon,whereby after a period of time there is a loss of the CRISPR enzymeexpression. In some aspects, one or more gRNA(s) are provided that arecapable of hybridization to one or more coding or non-coding regions ofthe polynucleotide encoding the CRISPR-Cas system, whereby after aperiod of time there is a inactivation of one or more, or in some casesall, of the CRISPR-Cas system. In some aspects of the system, and not tobe limited by theory, the cell may comprise a plurality of CRISPR-Cascomplexes, wherein a first subset of CRISPR complexes comprise a firstguide RNA capable of targeting a genomic locus or loci to be edited, anda second subset of CRISPR complexes comprise at least one second guideRNA capable of targeting the polynucleotide encoding the CRISPR-Cassystem, wherein the first subset of CRISPR-Cas complexes mediate editingof the targeted genomic locus or loci and the second subset of CRISPRcomplexes eventually inactivate the CRISPR-Cas system, therebyinactivating further CRISPR-Cas expression in the cell.

Thus the invention provides a CRISPR-Cas system comprising one or morevectors for delivery to a eukaryotic cell, wherein the vector(s)encode(s): (i) a CRISPR enzyme; (ii) a first guide RNA capable ofhybridizing to a target sequence in the cell; (iii) a second guide RNAcapable of hybridizing to one or more target sequence(s) in the vectorwhich encodes the CRISPR enzyme, when expressed within the cell: thefirst guide RNA directs sequence-specific binding of a first CRISPRcomplex to the target sequence in the cell; the second guide RNA directssequence-specific binding of a second CRISPR complex to the targetsequence in the vector which encodes the CRISPR enzyme; the CRISPRcomplexes comprise a CRISPR enzyme bound to a guide RNA, such that aguide RNA can hybridize to its target sequence; and the second CRISPRcomplex inactivates the CRISPR-Cas system to prevent continuedexpression of the CRISPR enzyme by the cell.

The various coding sequences (CRISPR enzyme and guide RNAs) can beincluded on a single vector or on multiple vectors. For instance, it ispossible to encode the enzyme on one vector and the various RNAsequences on another vector, or to encode the enzyme and one guide RNAon one vector, and the remaining guide RNA on another vector, or anyother permutation. In general, a system using a total of one or twodifferent vectors is preferred.

Where multiple vectors are used, it is possible to deliver them inunequal numbers, and ideally with an excess of a vector which encodesthe first guide RNA relative to the second guide RNA, thereby assistingin delaying final inactivation of the CRISPR system until genome editinghas had a chance to occur.

The first guide RNA can target any target sequence of interest within agenome, as described elsewhere herein. The second guide RNA targets asequence within the vector which encodes the CRISPR Cpf1 enzyme, andthereby inactivates the enzyme's expression from that vector. Thus thetarget sequence in the vector must be capable of inactivatingexpression. Suitable target sequences can be, for instance, near to orwithin the translational start codon for the Cpf1p coding sequence, in anon-coding sequence in the promoter driving expression of the non-codingRNA elements, within the promoter driving expression of the Cpf1p gene,within 100 bp of the ATG translational start codon in the Cas codingsequence, and/or within the inverted terminal repeat (iTR) of a viraldelivery vector, e.g., in the AAV genome. A double stranded break nearthis region can induce a frame shift in the Cas coding sequence, causinga loss of protein expression. An alternative target sequence for the“self-inactivating” guide RNA would aim to edit/inactivate regulatoryregions/sequences needed for the expression of the CRISPR-Cpf1 system orfor the stability of the vector. For instance, if the promoter for theCas coding sequence is disrupted then transcription can be inhibited orprevented. Similarly, if a vector includes sequences for replication,maintenance or stability then it is possible to target these. Forinstance, in a AAV vector a useful target sequence is within the iTR.Other useful sequences to target can be promoter sequences,polyadenlyation sites, etc.

Furthermore, if the guide RNAs are expressed in array format, the“self-inactivating” guide RNAs that target both promoters simultaneouslywill result in the excision of the intervening nucleotides from withinthe CRISPR-Cas expression construct, effectively leading to its completeinactivation. Similarly, excision of the intervening nucleotides willresult where the guide RNAs target both ITRs, or targets two or moreother CRISPR-Cas components simultaneously. Self-inactivation asexplained herein is applicable, in general, with CRISPR-Cas systems inorder to provide regulation of the CRISPR-Cas. For example,self-inactivation as explained herein may be applied to the CRISPRrepair of mutations, for example expansion disorders, as explainedherein. As a result of this self-inactivation, CRISPR repair is onlytransiently active.

Addition of non-targeting nucleotides to the 5′ end (e.g. 1-10nucleotides, preferably 1-5 nucleotides) of the “self-inactivating”guide RNA can be used to delay its processing and/or modify itsefficiency as a means of ensuring editing at the targeted genomic locusprior to CRISPR-Cas shutdown.

In one aspect of the self-inactivating AAV-CRISPR-Cas system, plasmidsthat co-express one or more guide RNA targeting genomic sequences ofinterest (e.g. 1-2, 1-5, 1-10, 1-15, 1-20, 1-30) may be established with“self-inactivating” guide RNAs that target an SpCas9 sequence at or nearthe engineered ATG start site (e.g. within 5 nucleotides, within 15nucleotides, within 30 nucleotides, within 50 nucleotides, within 100nucleotides). A regulatory sequence in the U6 promoter region can alsobe targeted with an guide RNA. The U6-driven guide RNAs may be designedin an array format such that multiple guide RNA sequences can besimultaneously released. When first delivered into target tissue/cells(left cell) guide RNAs begin to accumulate while Cas levels rise in thenucleus. Cas complexes with all of the guide RNAs to mediate genomeediting and self-inactivation of the CRISPR-Cas plasmids.

One aspect of a self-inactivating CRISPR-Cas system is expression ofsingly or in tandam array format from 1 up to 4 or more different guidesequences; e.g. up to about 20 or about 30 guides sequences. Eachindividual self inactivating guide sequence may target a differenttarget. Such may be processed from, e.g. one chimeric pol3 transcript.Pol3 promoters such as U6 or H1 promoters may be used. Pol2 promoterssuch as those mentioned throughout herein. Inverted terminal repeat(iTR) sequences may flank the Pol3 promoter-guide RNA(s)-Pol2promoter-Cas.

One aspect of a tandem array transcript is that one or more guide(s)edit the one or more target(s) while one or more self inactivatingguides inactivate the CRISPR-Cas system. Thus, for example, thedescribed CRISPR-Cas system for repairing expansion disorders may bedirectly combined with the self-inactivating CRISPR-Cas system describedherein. Such a system may, for example, have two guides directed to thetarget region for repair as well as at least a third guide directed toself-inactivation of the CRISPR-Cas. Reference is made to ApplicationSer. No. PCT/US2014/069897, entitled “Compositions And Methods Of Use OfCrispr-Cas Systems In Nucleotide Repeat Disorders,” published Dec. 12,2014 as WO/2015/089351.

The guideRNA may be a control guide. For example it may be engineered totarget a nucleic acid sequence encoding the CRISPR Enzyme itself, asdescribed in US2015232881A1, the disclosure of which is herebyincorporated by reference. In some embodiments, a system or compositionmay be provided with just the guideRNA engineered to target the nucleicacid sequence encoding the CRISPR Enzyme. In addition, the system orcomposition may be provided with the guideRNA engineered to target thenucleic acid sequence encoding the CRISPR Enzyme, as well as nucleicacid sequence encoding the CRISPR Enzyme and, optionally a second guideRNA and, further optionally, a repair template. The second guideRNA maybe the primary target of the CRISPR system or composition (such atherapeutic, diagnostic, knock out etc. as defined herein). In this way,the system or composition is self-inactivating. This is exemplified inrelation to Cas9 in US2015232881A1 (also published as WO2015070083 (A1)referenced elsewhere herein, and may be extrapolated to Cpf1.

Enzymes According to the Invention Used in a Multiplex (Tandem)Targeting Approach

The inventors have shown that CRISPR enzymes as defined herein canemploy more than one RNA guide without losing activity. This enables theuse of the CRISPR enzymes, systems or complexes as defined herein fortargeting multiple DNA targets, genes or gene loci, with a singleenzyme, system or complex as defined herein. The guide RNAs may betandemly arranged, optionally separated by a nucleotide sequence such asa direct repeat as defined herein. The position of the different guideRNAs is the tandem does not influence the activity. It is noted that theterms “CRISPR-Cas system”, “CRISP-Cas complex” “CRISPR complex” and“CRISPR system” are used interchangeably. Also the terms “CRISPRenzyme”, “Cas enzyme”, or “CRISPR-Cas enzyme”, can be usedinterchangeably. In preferred embodiments, said CRISPR enzyme, CRISP-Casenzyme or Cas enzyme is Cpf1, or any one of the modified or mutatedvariants thereof described herein elsewhere.

In one aspect, the invention provides a non-naturally occurring orengineered CRISPR enzyme, preferably a class 2 CRISPR enzyme, preferablya Type V or VI CRISPR enzyme as described herein, such as withoutlimitation Cpf1 as described herein elsewhere, used for tandem ormultiplex targeting. It is to be understood that any of the CRISPR (orCRISPR-Cas or Cas) enzymes, complexes, or systems according to theinvention as described herein elsewhere may be used in such an approach.Any of the methods, products, compositions and uses as described hereinelsewhere are equally applicable with the multiplex or tandem targetingapproach further detailed below. By means of further guidance, thefollowing particular aspects and embodiments are provided.

In one aspect, the invention provides for the use of a Cpf1 enzyme,complex or system as defined herein for targeting multiple gene loci. Inone embodiment, this can be established by using multiple (tandem ormultiplex) guide RNA (gRNA) sequences.

In one aspect, the invention provides methods for using one or moreelements of a Cpf1 enzyme, complex or system as defined herein fortandem or multiplex targeting, wherein said CRISP system comprisesmultiple guide RNA sequences. Preferably, said gRNA sequences areseparated by a nucleotide sequence, such as a direct repeat as definedherein elsewhere.

The Cpf1 enzyme, system or complex as defined herein provides aneffective means for modifying multiple target polynucleotides. The Cpf1enzyme, system or complex as defined herein has a wide variety ofutility including modifying (e.g., deleting, inserting, translocating,inactivating, activating) one or more target polynucleotides in amultiplicity of cell types. As such the Cpf1 enzyme, system or complexas defined herein of the invention has a broad spectrum of applicationsin, e.g., gene therapy, drug screening, disease diagnosis, andprognosis, including targeting multiple gene loci within a single CRISPRsystem.

In one aspect, the invention provides a Cpf1 enzyme, system or complexas defined herein, i.e. a Cpf1 CRISPR-Cas complex having a Cpf1 proteinhaving at least one destabilization domain associated therewith, andmultiple guide RNAs that target multiple nucleic acid molecules such asDNA molecules, whereby each of said multiple guide RNAs specificallytargets its corresponding nucleic acid molecule, e.g., DNA molecule.Each nucleic acid molecule target, e.g., DNA molecule can encode a geneproduct or encompass a gene locus. Using multiple guide RNAs henceenables the targeting of multiple gene loci or multiple genes. In someembodiments the Cpf1 enzyme may cleave the DNA molecule encoding thegene product. In some embodiments expression of the gene product isaltered. The Cpf1 protein and the guide RNAs do not naturally occurtogether. The invention comprehends the guide RNAs comprising tandemlyarranged guide sequences. The invention further comprehends codingsequences for the Cpf1 protein being codon optimized for expression in aeukaryotic cell. In a preferred embodiment the eukaryotic cell is amammalian cell, a plant cell or a yeast cell and in a more preferredembodiment the mammalian cell is a human cell. Expression of the geneproduct may be decreased. The Cpf1 enzyme may form part of a CRISPRsystem or complex, which further comprises tandemly arranged guide RNAs(gRNAs) comprising a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 25,30, or more than 30 guide sequences, each capable of specificallyhybridizing to a target sequence in a genomic locus of interest in acell. In some embodiments, the functional Cpf1 CRISPR system or complexbinds to the multiple target sequences. In some embodiments, thefunctional CRISPR system or complex may edit the multiple targetsequences, e.g., the target sequences may comprise a genomic locus, andin some embodiments there may be an alteration of gene expression. Insome embodiments, the functional CRISPR system or complex may comprisefurther functional domains. In some embodiments, the invention providesa method for altering or modifying expression of multiple gene products.The method may comprise introducing into a cell containing said targetnucleic acids, e.g., DNA molecules, or containing and expressing targetnucleic acid, e.g., DNA molecules; for instance, the target nucleicacids may encode gene products or provide for expression of geneproducts (e.g., regulatory sequences).

In preferred embodiments the CRISPR enzyme used for multiplex targetingis Cpf1, or the CRISPR system or complex comprises Cpf1. In someembodiments, the CRISPR enzyme used for multiplex targeting is AsCpf1,or the CRISPR system or complex used for multiplex targeting comprisesan AsCpf1. In some embodiments, the CRISPR enzyme is an LbCpf1, or theCRISPR system or complex comprises LbCpf1. In some embodiments, the Cpf1enzyme used for multiplex targeting cleaves both strands of DNA toproduce a double strand break (DSB). In some embodiments, the CRISPRenzyme used for multiplex targeting is a nickase. In some embodiments,the Cpf1 enzyme used for multiplex targeting is a dual nickase. In someembodiments, the Cpf1 enzyme used for multiplex targeting is a Cpf1enzyme such as a DD Cpf1 enzyme as defined herein elsewhere.

In some general embodiments, the Cpf1 enzyme used for multiplextargeting is associated with one or more functional domains. In somemore specific embodiments, the CRISPR enzyme used for multiplextargeting is a dead Cpf1 as defined herein elsewhere.

In an aspect, the present invention provides a means for delivering theCpf1 enzyme, system or complex for use in multiple targeting as definedherein or the polynucleotides defined herein. Non-limiting examples ofsuch delivery means are e.g. particle(s) delivering component(s) of thecomplex, vector(s) comprising the polynucleotide(s) discussed herein(e.g., encoding the CRISPR enzyme, providing the nucleotides encodingthe CRISPR complex). In some embodiments, the vector may be a plasmid ora viral vector such as AAV, or lentivirus. Transient transfection withplasmids, e.g., into HEK cells may be advantageous, especially given thesize limitations of AAV and that while Cpf1 fits into AAV, one may reachan upper limit with additional guide RNAs.

Also provided is a model that constitutively expresses the Cpf1 enzyme,complex or system as used herein for use in multiplex targeting. Theorganism may be transgenic and may have been transfected with thepresent vectors or may be the offspring of an organism so transfected.In a further aspect, the present invention provides compositionscomprising the CRISPR enzyme, system and complex as defined herein orthe polynucleotides or vectors described herein. Also provides are Cpf1CRISPR systems or complexes comprising multiple guide RNAs, preferablyin a tandemly arranged format. Said different guide RNAs may beseparated by nucleotide sequences such as direct repeats.

Also provided is a method of treating a subject, e.g., a subject in needthereof, comprising inducing gene editing by transforming the subjectwith the polynucleotide encoding the Cpf1 CRISPR system or complex orany of polynucleotides or vectors described herein and administeringthem to the subject. A suitable repair template may also be provided,for example delivered by a vector comprising said repair template. Alsoprovided is a method of treating a subject, e.g., a subject in needthereof, comprising inducing transcriptional activation or repression ofmultiple target gene loci by transforming the subject with thepolynucleotides or vectors described herein, wherein said polynucleotideor vector encodes or comprises the Cpf1 enzyme, complex or systemcomprising multiple guide RNAs, preferably tandemly arranged. Where anytreatment is occurring ex vivo, for example in a cell culture, then itwill be appreciated that the term ‘subject’ may be replaced by thephrase “cell or cell culture.”

Compositions comprising Cpf1 enzyme, complex or system comprisingmultiple guide RNAs, preferably tandemly arranged, or the polynucleotideor vector encoding or comprising said Cpf1 enzyme, complex or systemcomprising multiple guide RNAs, preferably tandemly arranged, for use inthe methods of treatment as defined herein elsewhere are also provided.A kit of parts may be provided including such compositions. Use of saidcomposition in the manufacture of a medicament for such methods oftreatment are also provided. Use of a Cpf1 CRISPR system in screening isalso provided by the present invention, e.g., gain of function screens.Cells which are artificially forced to overexpress a gene are be able todown regulate the gene over time (re-establishing equilibrium) e.g. bynegative feedback loops. By the time the screen starts the unregulatedgene might be reduced again. Using an inducible Cpf1 activator allowsone to induce transcription right before the screen and thereforeminimizes the chance of false negative hits. Accordingly, by use of theinstant invention in screening, e.g., gain of function screens, thechance of false negative results may be minimized.

In one aspect, the invention provides an engineered, non-naturallyoccurring CRISPR system comprising a Cpf1 protein and multiple guideRNAs that each specifically target a DNA molecule encoding a geneproduct in a cell, whereby the multiple guide RNAs each target theirspecific DNA molecule encoding the gene product and the Cpf1 proteincleaves the target DNA molecule encoding the gene product, wherebyexpression of the gene product is altered; and, wherein the CRISPRprotein and the guide RNAs do not naturally occur together. Theinvention comprehends the multiple guide RNAs comprising multiple guidesequences, preferably separated by a nucleotide sequence such as adirect repeat. In an embodiment of the invention the CRISPR protein is atype V or VI CRISPR-Cas protein and in a more preferred embodiment theCRIPSR protein is a Cpf1 protein. The invention further comprehends aCpf1 protein being codon optimized for expression in a eukaryotic cell.In a preferred embodiment the eukaryotic cell is a mammalian cell and ina more preferred embodiment the mammalian cell is a human cell. In afurther embodiment of the invention, the expression of the gene productis decreased.

In another aspect, the invention provides an engineered, non-naturallyoccurring vector system comprising one or more vectors comprising afirst regulatory element operably linked to the multiple Cpf1 CRISPRsystem guide RNAs that each specifically target a DNA molecule encodinga gene product and a second regulatory element operably linked codingfor a CRISPR protein. Both regulatory elements may be located on thesame vector or on different vectors of the system. The multiple guideRNAs target the multiple DNA molecules encoding the multiple geneproducts in a cell and the CRISPR protein may cleave the multiple DNAmolecules encoding the gene products (it may cleave one or both strandsor have substantially no nuclease activity), whereby expression of themultiple gene products is altered; and, wherein the CRISPR protein andthe multiple guide RNAs do not naturally occur together. In a preferredembodiment the CRISPR protein is Cpf1 protein, optionally codonoptimized for expression in a eukaryotic cell. In a preferred embodimentthe eukaryotic cell is a mammalian cell, a plant cell or a yeast celland in a more preferred embodiment the mammalian cell is a human cell.In a further embodiment of the invention, the expression of each of themultiple gene products is altered, preferably decreased.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the system comprises: (a) a firstregulatory element operably linked to a direct repeat sequence and oneor more insertion sites for inserting one or more guide sequences up- ordownstream (whichever applicable) of the direct repeat sequence, whereinwhen expressed, the one or more guide sequence(s) direct(s)sequence-specific binding of the CRISPR complex to the one or moretarget sequence(s) in a eukaryotic cell, wherein the CRISPR complexcomprises a Cpf1 enzyme complexed with the one or more guide sequence(s)that is hybridized to the one or more target sequence(s); and (b) asecond regulatory element operably linked to an enzyme-coding sequenceencoding said Cpf1 enzyme, preferably comprising at least one nuclearlocalization sequence and/or at least one NES; wherein components (a)and (b) are located on the same or different vectors of the system. Insome embodiments, component (a) further comprises two or more guidesequences operably linked to the first regulatory element, wherein whenexpressed, each of the two or more guide sequences direct sequencespecific binding of a Cpf1 CRISPR complex to a different target sequencein a eukaryotic cell. In some embodiments, the CRISPR complex comprisesone or more nuclear localization sequences and/or one or more NES ofsufficient strength to drive accumulation of said Cpf1 CRISPR complex ina detectable amount in or out of the nucleus of a eukaryotic cell. Insome embodiments, the first regulatory element is a polymerase IIIpromoter. In some embodiments, the second regulatory element is apolymerase II promoter. In some embodiments, each of the guide sequencesis at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, orbetween 16-25, or between 16-20 nucleotides in length.

Recombinant expression vectors can comprise the polynucleotides encodingthe Cpf1 enzyme, system or complex for use in multiple targeting asdefined herein in a form suitable for expression of the nucleic acid ina host cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors comprising the polynucleotidesencoding the Cpf1 enzyme, system or complex for use in multipletargeting as defined herein. In some embodiments, a cell is transfectedas it naturally occurs in a subject. In some embodiments, a cell that istransfected is taken from a subject. In some embodiments, the cell isderived from cells taken from a subject, such as a cell line. A widevariety of cell lines for tissue culture are known in the art andexemplidied herein elsewhere. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors comprising the polynucleotidesencoding the Cpf1 enzyme, system or complex for use in multipletargeting as defined herein is used to establish a new cell linecomprising one or more vector-derived sequences. In some embodiments, acell transiently transfected with the components of a Cpf1 CRISPR systemor complex for use in multiple targeting as described herein (such as bytransient transfection of one or more vectors, or transfection withRNA), and modified through the activity of a Cpf1 CRISPR system orcomplex, is used to establish a new cell line comprising cellscontaining the modification but lacking any other exogenous sequence. Insome embodiments, cells transiently or non-transiently transfected withone or more vectors comprising the polynucleotides encoding the Cpf1enzyme, system or complex for use in multiple targeting as definedherein, or cell lines derived from such cells are used in assessing oneor more test compounds.

The term “regulatory element” is as defined herein elsewhere.

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to a direct repeatsequence and one or more insertion sites for inserting one or more guideRNA sequences up- or downstream (whichever applicable) of the directrepeat sequence, wherein when expressed, the guide sequence(s) direct(s)sequence-specific binding of the Cpf1 CRISPR complex to the respectivetarget sequence(s) in a eukaryotic cell, wherein the Cpf1 CRISPR complexcomprises a Cpf1 enzyme complexed with the one or more guide sequence(s)that is hybridized to the respective target sequence(s); and/or (b) asecond regulatory element operably linked to an enzyme-coding sequenceencoding said Cpf1 enzyme comprising preferably at least one nuclearlocalization sequence and/or NES. In some embodiments, the host cellcomprises components (a) and (b). In some embodiments, component (a),component (b), or components (a) and (b) are stably integrated into agenome of the host eukaryotic cell. In some embodiments, component (a)further comprises two or more guide sequences operably linked to thefirst regulatory element, and optionally separated by a direct repeat,wherein when expressed, each of the two or more guide sequences directsequence specific binding of a Cpf1 CRISPR complex to a different targetsequence in a eukaryotic cell. In some embodiments, the Cpf1 enzymecomprises one or more nuclear localization sequences and/or nuclearexport sequences or NES of sufficient strength to drive accumulation ofsaid CRISPR enzyme in a detectable amount in and/or out of the nucleusof a eukaryotic cell.

In some embodiments, the Cpf1 enzyme is a type V or VI CRISPR systemenzyme. In some embodiments, the Cpf1 enzyme is a Cpf1 enzyme. In someembodiments, the Cpf1 enzyme is derived from Francisella tularensis 1,Francisella tularensis subsp. novicida, Prevotella albensis,Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus,Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacteriumGW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6,Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum,Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai,Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3,Prevotella disiens, or Porphyromonas macacae Cpf1, and may includefurther alterations or mutations of the Cpf1 as defined hereinelsewhere, and can be a chimeric Cpf1. In some embodiments, the Cpf1enzyme is codon-optimized for expression in a eukaryotic cell. In someembodiments, the CRISPR enzyme directs cleavage of one or two strands atthe location of the target sequence. In some embodiments, the firstregulatory element is a polymerase III promoter. In some embodiments,the second regulatory element is a polymerase II promoter. In someembodiments, the one or more guide sequence(s) is (are each) at least16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25,or between 16-20 nucleotides in length. When multiple guide RNAs areused, they are preferably separated by a direct repeat sequence. In anaspect, the invention provides a non-human eukaryotic organism;preferably a multicellular eukaryotic organism, comprising a eukaryotichost cell according to any of the described embodiments. In otheraspects, the invention provides a eukaryotic organism; preferably amulticellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. The organism in someembodiments of these aspects may be an animal; for example a mammal.Also, the organism may be an arthropod such as an insect. The organismalso may be a plant. Further, the organism may be a fungus.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises (a) a first regulatory element operablylinked to a direct repeat sequence and one or more insertion sites forinserting one or more guide sequences up- or downstream (whicheverapplicable) of the direct repeat sequence, wherein when expressed, theguide sequence directs sequence-specific binding of a Cpf1 CRISPRcomplex to a target sequence in a eukaryotic cell, wherein the Cpf1CRISPR complex comprises a Cpf1 enzyme complexed with the guide sequencethat is hybridized to the target sequence; and/or (b) a secondregulatory element operably linked to an enzyme-coding sequence encodingsaid Cpf1 enzyme comprising a nuclear localization sequence. In someembodiments, the kit comprises components (a) and (b) located on thesame or different vectors of the system. In some embodiments, component(a) further comprises two or more guide sequences operably linked to thefirst regulatory element, wherein when expressed, each of the two ormore guide sequences direct sequence specific binding of a CRISPRcomplex to a different target sequence in a eukaryotic cell. In someembodiments, the Cpf1 enzyme comprises one or more nuclear localizationsequences of sufficient strength to drive accumulation of said CRISPRenzyme in a detectable amount in the nucleus of a eukaryotic cell. Insome embodiments, the CRISPR enzyme is a type V or VI CRISPR systemenzyme. In some embodiments, the CRISPR enzyme is a Cpf1 enzyme. In someembodiments, the Cpf1 enzyme is derived from Francisella tularensis 1,Francisella tularensis subsp. novicida, Prevotella albensis,Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus,Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacteriumGW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6,Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum,Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai,Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3,Prevotella disiens, or Porphyromonas macacae Cpf1 (e.g., modified tohave or be associated with at least one DD), and may include furtheralteration or mutation of the Cpf1, and can be a chimeric Cpf1. In someembodiments, the DD-CRISPR enzyme is codon-optimized for expression in aeukaryotic cell. In some embodiments, the DD-CRISPR enzyme directscleavage of one or two strands at the location of the target sequence.In some embodiments, the DD-CRISPR enzyme lacks or substantially DNAstrand cleavage activity (e.g., no more than 5% nuclease activity ascompared with a wild type enzyme or enzyme not having the mutation oralteration that decreases nuclease activity). In some embodiments, thefirst regulatory element is a polymerase III promoter. In someembodiments, the second regulatory element is a polymerase II promoter.In some embodiments, the guide sequence is at least 16, 17, 18, 19, 20,25 nucleotides, or between 16-30, or between 16-25, or between 16-20nucleotides in length.

In one aspect, the invention provides a method of modifying multipletarget polynucleotides in a host cell such as a eukaryotic cell. In someembodiments, the method comprises allowing a Cpf1CRISPR complex to bindto multiple target polynucleotides, e.g., to effect cleavage of saidmultiple target polynucleotides, thereby modifying multiple targetpolynucleotides, wherein the Cpf1CRISPR complex comprises a Cpf1 enzymecomplexed with multiple guide sequences each of the being hybridized toa specific target sequence within said target polynucleotide, whereinsaid multiple guide sequences are linked to a direct repeat sequence. Insome embodiments, said cleavage comprises cleaving one or two strands atthe location of each of the target sequence by said Cpf1 enzyme. In someembodiments, said cleavage results in decreased transcription of themultiple target genes. In some embodiments, the method further comprisesrepairing one or more of said cleaved target polynucleotide byhomologous recombination with an exogenous template polynucleotide,wherein said repair results in a mutation comprising an insertion,deletion, or substitution of one or more nucleotides of one or more ofsaid target polynucleotides. In some embodiments, said mutation resultsin one or more amino acid changes in a protein expressed from a genecomprising one or more of the target sequence(s). In some embodiments,the method further comprises delivering one or more vectors to saideukaryotic cell, wherein the one or more vectors drive expression of oneor more of: the Cpf1 enzyme and the multiple guide RNA sequence linkedto a direct repeat sequence. In some embodiments, said vectors aredelivered to the eukaryotic cell in a subject. In some embodiments, saidmodifying takes place in said eukaryotic cell in a cell culture. In someembodiments, the method further comprises isolating said eukaryotic cellfrom a subject prior to said modifying. In some embodiments, the methodfurther comprises returning said eukaryotic cell and/or cells derivedtherefrom to said subject.

In one aspect, the invention provides a method of modifying expressionof multiple polynucleotides in a eukaryotic cell. In some embodiments,the method comprises allowing a Cpf1 CRISPR complex to bind to multiplepolynucleotides such that said binding results in increased or decreasedexpression of said polynucleotides; wherein the Cpf1 CRISPR complexcomprises a Cpf1 enzyme complexed with multiple guide sequences eachspecifically hybridized to its own target sequence within saidpolynucleotide, wherein said guide sequences are linked to a directrepeat sequence. In some embodiments, the method further comprisesdelivering one or more vectors to said eukaryotic cells, wherein the oneor more vectors drive expression of one or more of: the Cpf1 enzyme andthe multiple guide sequences linked to the direct repeat sequences.

In one aspect, the invention provides a recombinant polynucleotidecomprising multiple guide RNA sequences up- or downstream (whicheverapplicable) of a direct repeat sequence, wherein each of the guidesequences when expressed directs sequence-specific binding of aCpf1CRISPR complex to its corresponding target sequence present in aeukaryotic cell. In some embodiments, the target sequence is a viralsequence present in a eukaryotic cell. In some embodiments, the targetsequence is a proto-oncogene or an oncogene.

Aspects of the invention encompass a non-naturally occurring orengineered composition that may comprise a guide RNA (gRNA) comprising aguide sequence capable of hybridizing to a target sequence in a genomiclocus of interest in a cell and a Cpf1 enzyme as defined herein that maycomprise at least one or more nuclear localization sequences.

An aspect of the invention encompasses methods of modifying a genomiclocus of interest to change gene expression in a cell by introducinginto the cell any of the compositions described herein.

An aspect of the invention is that the above elements are comprised in asingle composition or comprised in individual compositions. Thesecompositions may advantageously be applied to a host to elicit afunctional effect on the genomic level.

As used herein, the term “guide RNA” or “gRNA” has the leaning as usedherein elsewhere and comprises any polynucleotide sequence havingsufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence. Each gRNA may be designed to includemultiple binding recognition sites (e.g., aptamers) specific to the sameor different adapter protein. Each gRNA may be designed to bind to thepromoter region −1000-+1 nucleic acids upstream of the transcriptionstart site (i.e. TSS), preferably −200 nucleic acids. This positioningimproves functional domains which affect gene activation (e.g.,transcription activators) or gene inhibition (e.g., transcriptionrepressors). The modified gRNA may be one or more modified gRNAstargeted to one or more target loci (e.g., at least 1 gRNA, at least 2gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 gRNA, at least 50 gRNA) comprised in a composition. Said multiple gRNAsequences can be tandemly arranged and are preferably separated by adirect repeat.

Thus, gRNA, the CRISPR enzyme as defined herein may each individually becomprised in a composition and administered to a host individually orcollectively. Alternatively, these components may be provided in asingle composition for administration to a host. Administration to ahost may be performed via viral vectors known to the skilled person ordescribed herein for delivery to a host (e.g., lentiviral vector,adenoviral vector, AAV vector). As explained herein, use of differentselection markers (e.g., for lentiviral gRNA selection) andconcentration of gRNA (e.g., dependent on whether multiple gRNAs areused) may be advantageous for eliciting an improved effect. On the basisof this concept, several variations are appropriate to elicit a genomiclocus event, including DNA cleavage, gene activation, or genedeactivation. Using the provided compositions, the person skilled in theart can advantageously and specifically target single or multiple lociwith the same or different functional domains to elicit one or moregenomic locus events. The compositions may be applied in a wide varietyof methods for screening in libraries in cells and functional modelingin vivo (e.g., gene activation of lincRNA and indentification offunction; gain-of-function modeling; loss-of-function modeling; the usethe compositions of the invention to establish cell lines and transgenicanimals for optimization and screening purposes).

The current invention comprehends the use of the compositions of thecurrent invention to establish and utilize conditional or inducibleCRISPR transgenic cell/animals; see, e.g., Platt et al., Cell (2014),159(2): 440-455, or PCT patent publications cited herein, such as WO2014/093622 (PCT/US2013/074667). For example, cells or animals such asnon-human animals, e.g., vertebrates or mammals, such as rodents, e.g.,mice, rats, or other laboratory or field animals, e.g., cats, dogs,sheep, etc., may be ‘knock-in’ whereby the animal conditionally orinducibly expresses Cpf1 akin to Platt et al. The target cell or animalthus comprises the CRISRP enzyme (e.g., Cpf1) conditionally or inducibly(e.g., in the form of Cre dependent constructs), on expression of avector introduced into the target cell, the vector expresses that whichinduces or gives rise to the condition of the CRISRP enzyme (e.g., Cpf1)expression in the target cell. By applying the teaching and compositionsas defined herein with the known method of creating a CRISPR complex,inducible genomic events are also an aspect of the current invention.Examples of such inducible events have been described herein elsewhere.

In some embodiments, phenotypic alteration is preferably the result ofgenome modification when a genetic disease is targeted, especially inmethods of therapy and preferably where a repair template is provided tocorrect or alter the phenotype.

In some embodiments diseases that may be targeted include thoseconcerned with disease-causing splice defects.

In some embodiments, cellular targets include HemopoieticStem/Progenitor Cells (CD34+); Human T cells; and Eye (retinalcells)—for example photoreceptor precursor cells.

In some embodiments Gene targets include: Human Beta Globin—BBB (fortreating Sickle Cell Anemia, including by stimulating gene-conversion(using closely related HBD gene as an endogenous template)); CD3(T-Cells); and CEP920—retina (eye).

In some embodiments disease targets also include: cancer; Sickle CellAnemia (based on a point mutation); HBV, HIV; Beta-Thalassemia; andophthalmic or ocular disease—for example Leber Congenital Amaurosis(LCA)-causing Splice Defect.

In some embodiments delivery methods include: Cationic Lipid Mediated“direct” delivery of Enzyme-Guide complex (RiboNucleoProtein) andelectroporation of plasmid DNA.

Methods, products and uses described herein may be used fornon-therapeutic purposes. Furthermore, any of the methods describedherein may be applied in vitro and ex vivo.

In an aspect, provided is a non-naturally occurring or engineeredcomposition comprising:

I. two or more CRISPR-Cas system polynucleotide sequences comprising

(a) a first guide sequence capable of hybridizing to a first targetsequence in a polynucleotide locus,

(b) a second guide sequence capable of hybridizing to a second targetsequence in a polynucleotide locus,

(c) a direct repeat sequence,

and

II. a Cpf1 enzyme or a second polynucleotide sequence encoding it,

wherein when transcribed, the first and the second guide sequencesdirect sequence-specific binding of a first and a second Cpf1 CRISPRcomplex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the Cpf1 enzyme complexedwith the first guide sequence that is hybridizable to the first targetsequence,

wherein the second CRISPR complex comprises the Cpf1 enzyme complexedwith the second guide sequence that is hybridizable to the second targetsequence, and

wherein the first guide sequence directs cleavage of one strand of theDNA duplex near the first target sequence and the second guide sequencedirects cleavage of the other strand near the second target sequenceinducing a double strand break, thereby modifying the organism or thenon-human or non-animal organism. Similarly, compositions comprisingmore than two guide RNAs can be envisaged e.g. each specific for onetarget, and arranged tandemly in the composition or CRISPR system orcomplex as described herein.

In another embodiment, the Cpf1 is delivered into the cell as a protein.In another and particularly preferred embodiment, the Cpf1 is deliveredinto the cell as a protein or as a nucleotide sequence encoding it.Delivery to the cell as a protein may include delivery of aRibonucleoprotein (RNP) complex, where the protein is complexed with themultiple guides.

In an aspect, host cells and cell lines modified by or comprising thecompositions, systems or modified enzymes of present invention areprovided, including stem cells, and progeny thereof.

In an aspect, methods of cellular therapy are provided, where, forexample, a single cell or a population of cells is sampled or cultured,wherein that cell or cells is or has been modified ex vivo as describedherein, and is then re-introduced (sampled cells) or introduced(cultured cells) into the organism. Stem cells, whether embryonic orinduce pluripotent or totipotent stem cells, are also particularlypreferred in this regard. But, of course, in vivo embodiments are alsoenvisaged.

Inventive methods can further comprise delivery of templates, such asrepair templates, which may be dsODN or ssODN, see below. Delivery oftemplates may be via the cotemporaneous or separate from delivery of anyor all the CRISPR enzyme or guide RNAs and via the same deliverymechanism or different. In some embodiments, it is preferred that thetemplate is delivered together with the guide RNAs and, preferably, alsothe CRISPR enzyme. An example may be an AAV vector where the CRISPRenzyme is AsCpf1 or LbCpf1.

Inventive methods can further comprise: (a) delivering to the cell adouble-stranded oligodeoxynucleotide (dsODN) comprising overhangscomplimentary to the overhangs created by said double strand break,wherein said dsODN is integrated into the locus of interest; or —(b)delivering to the cell a single-stranded oligodeoxynucleotide (ssODN),wherein said ssODN acts as a template for homology directed repair ofsaid double strand break. Inventive methods can be for the prevention ortreatment of disease in an individual, optionally wherein said diseaseis caused by a defect in said locus of interest. Inventive methods canbe conducted in vivo in the individual or ex vivo on a cell taken fromthe individual, optionally wherein said cell is returned to theindividual.

The invention also comprehends products obtained from using CRISPRenzyme or Cas enzyme or Cpf1 enzyme or CRISPR-CRISPR enzyme orCRISPR-Cas system or CRISPR-Cpf1 system for use in tandem or multipletargeting as defined herein.

Kits

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises a vector system as taught herein andinstructions for using the kit. Elements may be provided individually orin combinations, and may be provided in any suitable container, such asa vial, a bottle, or a tube. The kits may include the gRNA and theunbound protector strand as described herein. The kits may include thegRNA with the protector strand bound to at least partially to the guidesequence (i.e. pgRNA). Thus the kits may include the pgRNA in the formof a partially double stranded nucleotide sequence as described here. Insome embodiments, the kit includes instructions in one or morelanguages, for example in more than one language. The instructions maybe specific to the applications and methods described herein.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.,in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide. In some embodiments, the kitcomprises one or more of the vectors and/or one or more of thepolynucleotides described herein. The kit may advantageously allows toprovide all elements of the systems of the invention.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utility includingmodifying (e.g., deleting, inserting, translocating, inactivating,activating) a target polynucleotide in a multiplicity of cell types. Assuch the CRISPR complex of the invention has a broad spectrum ofapplications in, e.g., gene therapy, drug screening, disease diagnosis,and prognosis. An exemplary CRISPR complex comprises a CRISPR effectorprotein complexed with a guide sequence hybridized to a target sequencewithin the target polynucleotide. In certain embodiments, a directrepeat sequence is linked to the guide sequence.

In one embodiment, this invention provides a method of cleaving a targetpolynucleotide. The method comprises modifying a target polynucleotideusing a CRISPR complex that binds to the target polynucleotide andeffect cleavage of said target polynucleotide. Typically, the CRISPRcomplex of the invention, when introduced into a cell, creates a break(e.g., a single or a double strand break) in the genome sequence. Forexample, the method can be used to cleave a disease gene in a cell.

The break created by the CRISPR complex can be repaired by a repairprocesses such as the error prone non-homologous end joining (NHEJ)pathway or the high fidelity homology directed repair (HDR). Duringthese repair process, an exogenous polynucleotide template can beintroduced into the genome sequence. In some methods, the HDR process isused to modify genome sequence. For example, an exogenous polynucleotidetemplate comprising a sequence to be integrated flanked by an upstreamsequence and a downstream sequence is introduced into a cell. Theupstream and downstream sequences share sequence similarity with eitherside of the site of integration in the chromosome.

Where desired, a donor polynucleotide can be DNA, e.g., a DNA plasmid, abacterial artificial chromosome (BAC), a yeast artificial chromosome(YAC), a viral vector, a linear piece of DNA, a PCR fragment, a nakednucleic acid, or a nucleic acid complexed with a delivery vehicle suchas a liposome or poloxamer.

The exogenous polynucleotide template comprises a sequence to beintegrated (e.g., a mutated gene). The sequence for integration may be asequence endogenous or exogenous to the cell. Examples of a sequence tobe integrated include polynucleotides encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction.

The upstream and downstream sequences in the exogenous polynucleotidetemplate are selected to promote recombination between the chromosomalsequence of interest and the donor polynucleotide. The upstream sequenceis a nucleic acid sequence that shares sequence similarity with thegenome sequence upstream of the targeted site for integration.Similarly, the downstream sequence is a nucleic acid sequence thatshares sequence similarity with the chromosomal sequence downstream ofthe targeted site of integration. The upstream and downstream sequencesin the exogenous polynucleotide template can have 75%, 80%, 85%, 90%,95%, or 100% sequence identity with the targeted genome sequence.Preferably, the upstream and downstream sequences in the exogenouspolynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100%sequence identity with the targeted genome sequence. In some methods,the upstream and downstream sequences in the exogenous polynucleotidetemplate have about 99% or 100% sequence identity with the targetedgenome sequence.

An upstream or downstream sequence may comprise from about 20 bp toabout 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplaryupstream or downstream sequence have about 200 bp to about 2000 bp,about 600 bp to about 1000 bp, or more particularly about 700 bp toabout 1000 bp.

In some methods, the exogenous polynucleotide template may furthercomprise a marker. Such a marker may make it easy to screen for targetedintegrations. Examples of suitable markers include restriction sites,fluorescent proteins, or selectable markers. The exogenouspolynucleotide template of the invention can be constructed usingrecombinant techniques (see, for example, Sambrook et al., 2001 andAusubel et al., 1996).

In an exemplary method for modifying a target polynucleotide byintegrating an exogenous polynucleotide template, a double strandedbreak is introduced into the genome sequence by the CRISPR complex, thebreak is repaired via homologous recombination an exogenouspolynucleotide template such that the template is integrated into thegenome. The presence of a double-stranded break facilitates integrationof the template.

In other embodiments, this invention provides a method of modifyingexpression of a polynucleotide in a eukaryotic cell. The methodcomprises increasing or decreasing expression of a target polynucleotideby using a CRISPR complex that binds to the polynucleotide.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced.

In some methods, a control sequence can be inactivated such that it nolonger functions as a control sequence. As used herein, “controlsequence” refers to any nucleic acid sequence that effects thetranscription, translation, or accessibility of a nucleic acid sequence.Examples of a control sequence include, a promoter, a transcriptionterminator, and an enhancer are control sequences. The inactivatedtarget sequence may include a deletion mutation (i.e., deletion of oneor more nucleotides), an insertion mutation (i.e., insertion of one ormore nucleotides), or a nonsense mutation (i.e., substitution of asingle nucleotide for another nucleotide such that a stop codon isintroduced). In some methods, the inactivation of a target sequenceresults in “knockout” of the target sequence.

Exemplary Methods of Using of CRISPR Cas System

The invention provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in amodifying a target cell in vivo, ex vivo or in vitro and, may beconducted in a manner alters the cell such that once modified theprogeny or cell line of the CRISPR modified cell retains the alteredphenotype. The modified cells and progeny may be part of amulti-cellular organism such as a plant or animal with ex vivo or invivo application of CRISPR system to desired cell types. The CRISPRinvention may be a therapeutic method of treatment. The therapeuticmethod of treatment may comprise gene or genome editing, or genetherapy.

Use of Inactivated CRISPR Cpf1 Enzyme for Detection Methods Such as FISH

In one aspect, the invention provides an engineered, non-naturallyoccurring CRISPR-Cas system comprising a catalytically inactivate Casprotein described herein, preferably an inactivate Cpf1 (dCpf1), and usethis system in detection methods such as fluorescence in situhybridization (FISH). dCpf1 which lacks the ability to produce DNAdouble-strand breaks may be fused with a marker, such as fluorescentprotein, such as the enhanced green fluorescent protein (eEGFP) andco-expressed with small guide RNAs to target pericentric, centric andteleomeric repeats in vivo. The dCpf1 system can be used to visualizeboth repetitive sequences and individual genes in the human genome. Suchnew applications of labelled dCpf1 CRISPR-cas systems may be importantin imaging cells and studying the functional nuclear architecture,especially in cases with a small nucleus volume or complex 3-Dstructures. (Chen B, Gilbert L A, Cimini B A, Schnitzbauer J, Zhang W,Li G W, Park J, Blackburn E H, Weissman J S, Qi L S, Huang B. 2013.Dynamic imaging of genomic loci in living human cells by an optimizedCRISPR/Cas system. Cell 155(7):1479-91. doi: 10.1016/j.cell.2013.12.001)

Modifying a Target with CRISPR Cas System or Complex (e.g., Cpf1-RNAComplex)

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal, and modifying thecell or cells. Culturing may occur at any stage ex vivo. The cell orcells may even be re-introduced into the non-human animal or plant. Forre-introduced cells it is particularly preferred that the cells are stemcells.

In some embodiments, the method comprises allowing a CRISPR complex tobind to the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR enzyme complexed with a guide sequencehybridized or hybridizable to a target sequence within said targetpolynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized or hybridizable to atarget sequence within said polynucleotide. Similar considerations andconditions apply as above for methods of modifying a targetpolynucleotide. In fact, these sampling, culturing and re-introductionoptions apply across the aspects of the present invention.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR enzyme complexed with a guide sequence hybridized orhybridizable to a target sequence. Similar considerations and conditionsapply as above for methods of modifying a target polynucleotide.

Thus in any of the non-naturally-occurring CRISPR enzymes describedherein comprise at least one modification and whereby the enzyme hascertain improved capabilities. In particular, any of the enzymes arecapable of forming a CRISPR complex with a guide RNA. When such acomplex forms, the guide RNA is capable of binding to a targetpolynucleotide sequence and the enzyme is capable of modifying a targetlocus. In addition, the enzyme in the CRISPR complex has reducedcapability of modifying one or more off-target loci as compared to anunmodified enzyme.

In addition, the modified CRISPR enzymes described herein encompassenzymes whereby in the CRISPR complex the enzyme has increasedcapability of modifying the one or more target loci as compared to anunmodified enzyme. Such function may be provided separate to or providedin combination with the above-described function of reduced capabilityof modifying one or more off-target loci. Any such enzymes may beprovided with any of the further modifications to the CRISPR enzyme asdescribed herein, such as in combination with any activity provided byone or more associated heterologous functional domains, any furthermutations to reduce nuclease activity and the like.

In advantageous embodiments of the invention, the modified CRISPR enzymeis provided with reduced capability of modifying one or more off-targetloci as compared to an unmodified enzyme and increased capability ofmodifying the one or more target loci as compared to an unmodifiedenzyme. In combination with further modifications to the enzyme,significantly enhanced specificity may be achieved. For example,combination of such advantageous embodiments with one or more additionalmutations is provided wherein the one or more additional mutations arein one or more catalytically active domains. Such further catalyticmutations may confer nickase functionality as described in detailelsewhere herein. In such enzymes, enhanced specificity may be achieveddue to an improved specificity in terms of enzyme activity.

Modifications to reduce off-target effects and/or enhance on-targeteffects as described above may be made to amino acid residues located ina positively-charged region/groove situated between the RuvC-III and HNHdomains. It will be appreciated that any of the functional effectsdescribed above may be achieved by modification of amino acids withinthe aforementioned groove but also by modification of amino acidsadjacent to or outside of that groove.

Additional functionalities which may be engineered into modified CRISPRenzymes as described herein include the following. 1. modified CRISPRenzymes that disrupt DNA:protein interactions without affecting proteintertiary or secondary structure. This includes residues that contact anypart of the RNA:DNA duplex. 2. modified CRISPR enzymes that weakenintra-protein interactions holding Cpf1 in conformation essential fornuclease cutting in response to DNA binding (on or off target). Forexample: a modification that mildly inhibits, but still allows, thenuclease conformation of the HNH domain (positioned at the scissilephosphate). 3. modified CRISPR enzymes that strengthen intra-proteininteractions holding Cpf1 in a conformation inhibiting nuclease activityin response to DNA binding (on or off targets). For example: amodification that stabilizes the HNH domain in a conformation away fromthe scissile phosphate. Any such additional functional enhancement maybe provided in combination with any other modification to the CRISPRenzyme as described in detail elsewhere herein.

Any of the herein described improved functionalities may be made to anyCRISPR enzyme, such as a Cpf1 enzyme. However, it will be appreciatedthat any of the functionalities described herein may be engineered intoCpf1 enzymes from other orthologs, including chimeric enzymes comprisingfragments from multiple orthologs.

Nucleic Acids, Amino Acids and Proteins, Regulatory Sequences, Vectors,Etc.

The invention uses nucleic acids to bind target DNA sequences. This isadvantageous as nucleic acids are much easier and cheaper to producethan proteins, and the specificity can be varied according to the lengthof the stretch where homology is sought. Complex 3-D positioning ofmultiple fingers, for example is not required. The terms“polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid”and “oligonucleotide” are used interchangeably. They refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Polynucleotides may have anythree dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridising to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 25° C. lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 15° C. lowerthan the T_(m). In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30° C. lower than the T_(m).Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife—eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences.

In aspects of the invention the term “guide RNA”, refers to thepolynucleotide sequence comprising a putative or identified crRNAsequence or guide sequence.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature. In all aspectsand embodiments, whether they include these terms or not, it will beunderstood that, preferably, the may be optional and thus preferablyincluded or not preferably not included. Furthermore, the terms“non-naturally occurring” and “engineered” may be used interchangeablyand so can therefore be used alone or in combination and one or othermay replace mention of both together. In particular, “engineered” ispreferred in place of “non-naturally occurring” or “non-naturallyoccurring and/or engineered.”

Sequence homologies may be generated by any of a number of computerprograms known in the art, for example BLAST or FASTA, etc. A suitablecomputer program for carrying out such an alignment is the GCG WisconsinBestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984,Nucleic Acids Research 12:387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). Howeverit is preferred to use the GCG Bestfit program. Percentage (%) sequencehomology may be calculated over contiguous sequences, i.e., one sequenceis aligned with the other sequence and each amino acid or nucleotide inone sequence is directly compared with the corresponding amino acid ornucleotide in the other sequence, one residue at a time. This is calledan “ungapped” alignment. Typically, such ungapped alignments areperformed only over a relatively short number of residues. Although thisis a very simple and consistent method, it fails to take intoconsideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion may cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without unduly penalizing the overall homology or identityscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximize local homology or identity. However, these more complexmethods assign “gap penalties” to each gap that occurs in the alignmentso that, for the same number of identical amino acids, a sequencealignment with as few gaps as possible—reflecting higher relatednessbetween the two compared sequences—may achieve a higher score than onewith many gaps. “Affinity gap costs” are typically used that charge arelatively high cost for the existence of a gap and a smaller penaltyfor each subsequent residue in the gap. This is the most commonly usedgap scoring system. High gap penalties may, of course, produce optimizedalignments with fewer gaps. Most alignment programs allow the gappenalties to be modified. However, it is preferred to use the defaultvalues when using such software for sequence comparisons. For example,when using the GCG Wisconsin Bestfit package the default gap penalty foramino acid sequences is −12 for a gap and −4 for each extension.Calculation of maximum % homology therefore first requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed.—Chapter 18), FASTA (Altschul et al., 1990 J Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

Set Sub-set Hydrophobic F W Y H K M I L V Aromatic F W Y H A G CAliphatic I L V Polar W Y H K R E D C S Charged H K R E D T N QPositively H K R charged Negatively E D charged Small V C A G S P T N DTiny A G S

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyriylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or β-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the α-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid Form are Known in the Art, for ExampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

Homology modelling: Corresponding residues in other Cpf1 orthologs canbe identified by the methods of Zhang et al., 2012 (Nature; 490(7421):556-60) and Chen et al., 2015 (PLoS Comput Biol; 11(5): e1004248)—acomputational protein-protein interaction (PPI) method to predictinteractions mediated by domain-motif interfaces. PrePPI (PredictingPPI), a structure based PPI prediction method, combines structuralevidence with non-structural evidence using a Bayesian statisticalframework. The method involves taking a pair a query proteins and usingstructural alignment to identify structural representatives thatcorrespond to either their experimentally determined structures orhomology models. Structural alignment is further used to identify bothclose and remote structural neighbours by considering global and localgeometric relationships. Whenever two neighbors of the structuralrepresentatives form a complex reported in the Protein Data Bank, thisdefines a template for modelling the interaction between the two queryproteins. Models of the complex are created by superimposing therepresentative structures on their corresponding structural neighbour inthe template. This approach is further described in Dey et al., 2013(Prot Sci; 22: 359-66).

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g. circular); nucleic acid molecules that compriseDNA, RNA, or both; and other varieties of polynucleotides known in theart. One type of vector is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beinserted, such as by standard molecular cloning techniques. Another typeof vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g. bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for guide RNA and(optionally modified or mutated) CRISPR enzymes (e.g. Cpf1). Bicistronicexpression vectors for guide RNA and (optionally modified or mutated)CRISPR enzymes are preferred. In general and particularly in thisembodiment (optionally modified or mutated) CRISPR enzymes arepreferably driven by the CBh promoter. The RNA may preferably be drivenby a Pol III promoter, such as a U6 promoter. Ideally the two arecombined.

In some embodiments, a loop in the guide RNA is provided. This may be astem loop or a tetra loop. The loop is preferably GAAA, but it is notlimited to this sequence or indeed to being only 4 bp in length. Indeed,preferred loop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG. Inpracticing any of the methods disclosed herein, a suitable vector can beintroduced to a cell or an embryo via one or more methods known in theart, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g. transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g. 1, 2,3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g.1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters(e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.Examples of pol III promoters include, but are not limited to, U6 and H1promoters. Examples of pol II promoters include, but are not limited to,the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally withthe RSV enhancer), the cytomegalovirus (CMV) promoter (optionally withthe CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter. Also encompassed by the term “regulatory element” are enhancerelements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR ofHTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer;and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc.Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression desired, etc. A vectorcan be introduced into host cells to thereby produce transcripts,proteins, or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., clustered regularlyinterspersed short palindromic repeats (CRISPR) transcripts, proteins,enzymes, mutant forms thereof, fusion proteins thereof, etc.). Withregards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g. amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amrann etal., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 60-89). In some embodiments, a vector is a yeastexpression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.). In some embodiments, a vector drives protein expression ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety. In some embodiments, a regulatoryelement is operably linked to one or more elements of a CRISPR system soas to drive expression of the one or more elements of the CRISPR system.In general, CRISPRs (Clustered Regularly Interspaced Short PalindromicRepeats), also known as SPIDRs (SPacer Interspersed Direct Repeats),constitute a family of DNA loci that are usually specific to aparticular bacterial species. The CRISPR locus comprises a distinctclass of interspersed short sequence repeats (SSRs) that were recognizedin E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; andNakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associatedgenes. Similar interspersed SSRs have been identified in Haloferaxmediterranei, Streptococcus pyogenes, Anabaena, and Mycobacteriumtuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993];Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al.,Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol.Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ fromother SSRs by the structure of the repeats, which have been termed shortregularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol.,6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).In general, the repeats are short elements that occur in clusters thatare regularly spaced by unique intervening sequences with asubstantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “nucleic acid-targeting system” as used in the presentapplication refers collectively to transcripts and other elementsinvolved in the expression of or directing the activity of nucleicacid-targeting CRISPR-associated (“Cas”) genes (also referred to hereinas an effector protein), including sequences encoding a nucleicacid-targeting Cas (effector) protein and a guide RNA or other sequencesand transcripts from a nucleic acid-targeting CRISPR locus. In someembodiments, one or more elements of a nucleic acid-targeting system arederived from a Type V/Type VI nucleic acid-targeting CRISPR system. Insome embodiments, one or more elements of a nucleic acid-targetingsystem is derived from a particular organism comprising an endogenousnucleic acid-targeting CRISPR system. In general, a nucleicacid-targeting system is characterized by elements that promote theformation of a nucleic acid-targeting complex at the site of a targetsequence. In the context of formation of a nucleic acid-targetingcomplex, “target sequence” refers to a sequence to which a guidesequence is designed to have complementarity, where hybridizationbetween a target sequence and a guide RNA promotes the formation of aDNA or RNA-targeting complex. Full complementarity is not necessarilyrequired, provided there is sufficient complementarity to causehybridization and promote formation of a nucleic acid-targeting complex.A target sequence may comprise RNA polynucleotides. In some embodiments,a target sequence is located in the nucleus or cytoplasm of a cell. Insome embodiments, the target sequence may be within an organelle of aeukaryotic cell, for example, mitochondrion or chloroplast. A sequenceor template that may be used for recombination into the targeted locuscomprising the target sequences is referred to as an “editing template”or “editing RNA” or “editing sequence”. In aspects of the invention, anexogenous template RNA may be referred to as an editing template. In anaspect of the invention the recombination is homologous recombination.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth RNA strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, or more base pairs from) the target sequence. In someembodiments, one or more vectors driving expression of one or moreelements of a nucleic acid-targeting system are introduced into a hostcell such that expression of the elements of the nucleic acid-targetingsystem direct formation of a nucleic acid-targeting complex at one ormore target sites. For example, a nucleic acid-targeting effectorprotein and a guide RNA could each be operably linked to separateregulatory elements on separate vectors. Alternatively, two or more ofthe elements expressed from the same or different regulatory elements,may be combined in a single vector, with one or more additional vectorsproviding any components of the nucleic acid-targeting system notincluded in the first vector. nucleic acid-targeting system elementsthat are combined in a single vector may be arranged in any suitableorientation, such as one element located 5′ with respect to (“upstream”of) or 3′ with respect to (“downstream” of) a second element. The codingsequence of one element may be located on the same or opposite strand ofthe coding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a nucleic acid-targeting effectorprotein and a guide RNA embedded within one or more intron sequences(e.g. each in a different intron, two or more in at least one intron, orall in a single intron). In some embodiments, the nucleic acid-targetingeffector protein and guide RNA are operably linked to and expressed fromthe same promoter.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a nucleic acid-targeting complex to the target sequence. In someembodiments, the degree of complementarity between a guide sequence andits corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, SanDiego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. The ability of a guide sequence to directsequence-specific binding of a nucleic acid-targeting complex to atarget sequence may be assessed by any suitable assay. For example, thecomponents of a nucleic acid-targeting system sufficient to form anucleic acid-targeting complex, including the guide sequence to betested, may be provided to a host cell having the corresponding targetsequence, such as by transfection with vectors encoding the componentsof the nucleic acid-targeting CRISPR sequence, followed by an assessmentof preferential cleavage within or in the vicinity of the targetsequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target polynucleotide sequence (or a sequence in thevicinity thereof) may be evaluated in a test tube by providing thetarget sequence, components of a nucleic acid-targeting complex,including the guide sequence to be tested and a control guide sequencedifferent from the test guide sequence, and comparing binding or rate ofcleavage at or in the vicinity of the target sequence between the testand control guide sequence reactions. Other assays are possible, andwill occur to those skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a gene transcriptor mRNA.

In some embodiments, the target sequence is a sequence within a genomeof a cell.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSer. No. TBA (attorney docket 44790.11.2022; Broad ReferenceBI-2013/004A); incorporated herein by reference.

In some embodiments, a recombination template is also provided. Arecombination template may be a component of another vector as describedherein, contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by a nucleicacid-targeting effector protein as a part of a nucleic acid-targetingcomplex. A template polynucleotide may be of any suitable length, suchas about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500,1000, or more nucleotides in length. In some embodiments, the templatepolynucleotide is complementary to a portion of a polynucleotidecomprising the target sequence. When optimally aligned, a templatepolynucleotide might overlap with one or more nucleotides of a targetsequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In someembodiments, when a template sequence and a polynucleotide comprising atarget sequence are optimally aligned, the nearest nucleotide of thetemplate polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75,100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from thetarget sequence.

In some embodiments, the nucleic acid-targeting effector protein is partof a fusion protein comprising one or more heterologous protein domains(e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredomains in addition to the nucleic acid-targeting effector protein). Insome embodiments, the CRISPR effector protein is part of a fusionprotein comprising one or more heterologous protein domains (e.g. aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains inaddition to the CRISPR enzyme). A CRISPR enzyme fusion protein maycomprise any additional protein sequence, and optionally a linkersequence between any two domains. Examples of protein domains that maybe fused to a CRISPR enzyme include, without limitation, epitope tags,reporter gene sequences, and protein domains having one or more of thefollowing activities: methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,RNA cleavage activity and nucleic acid binding activity. Non-limitingexamples of epitope tags include histidine (His) tags, V5 tags, FLAGtags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, andthioredoxin (Trx) tags. Examples of reporter genes include, but are notlimited to, glutathione-S-transferase (GST), horseradish peroxidase(HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP). ACRISPR enzyme may be fused to a gene sequence encoding a protein or afragment of a protein that bind DNA molecules or bind other cellularmolecules, including but not limited to maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domainfusions, and herpes simplex virus (HSV) BP16 protein fusions. Additionaldomains that may form part of a fusion protein comprising a CRISPRenzyme are described in US20110059502, incorporated herein by reference.In some embodiments, a tagged CRISPR enzyme is used to identify thelocation of a target sequence.

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283 and WO 2014/018423 andU.S. Pat. Nos. 8,889,418, 8,895,308, US20140186919, US20140242700,US20140273234, US20140335620, WO2014093635, which is hereby incorporatedby reference in its entirety.

Delivery

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a nucleic acid-targeting effector protein incombination with (and optionally complexed with) a guide RNA isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a nucleic acid-targeting system to cells inculture, or in a host organism. Non-viral vector delivery systemsinclude DNA plasmids, RNA (e.g. a transcript of a vector describedherein), naked nucleic acid, and nucleic acid complexed with a deliveryvehicle, such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Options for DNA/RNA or DNA/DNA or RNA/RNA or Protein/RNA

In some embodiments, the components of the CRISPR system may bedelivered in various form, such as combinations of DNA/RNA or RNA/RNA orprotein RNA. For example, the Cpf1 may be delivered as a DNA-codingpolynucleotide or an RNA-coding polynucleotide or as a protein. Theguide may be delivered may be delivered as a DNA-coding polynucleotideor an RNA. All possible combinations are envisioned, including mixedforms of delivery.

In some embodiments, all such combinations (DNA/RNA or DNA/DNA orRNA/RNA or protein/RNA).

In some embodiment, when the Cpf1 is delivered in protein form, it ispossible to pre-assemble same with one or more guide/s.

Nanoclews

Further, the CRISPR system may be delivered using nanoclews, for exampleas described in Sun W et al, Cocoon-like self-degradable DNA nanoclewfor anticancer drug delivery., J Am Chem Soc. 2014 Oct. 22;136(42):14722-5. doi: 10.1021/ja5088024. Epub 2014 Oct. 13; or in Sun Wet al, Self-Assembled DNA Nanoclews for the Efficient Delivery ofCRISPR-Cas9 for Genome Editing., Angew Chem Int Ed Engl. 2015 Oct. 5;54(41):12029-33. doi: 10.1002/anie.201506030. Epub 2015 Aug. 27.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Models of Genetic and Epigenetic Conditions

A method of the invention may be used to create a plant, an animal orcell that may be used to model and/or study genetic or epigeneticconditions of interest, such as a through a model of mutations ofinterest or a disease model. As used herein, “disease” refers to adisease, disorder, or indication in a subject. For example, a method ofthe invention may be used to create an animal or cell that comprises amodification in one or more nucleic acid sequences associated with adisease, or a plant, animal or cell in which the expression of one ormore nucleic acid sequences associated with a disease are altered. Sucha nucleic acid sequence may encode a disease associated protein sequenceor may be a disease associated control sequence. Accordingly, it isunderstood that in embodiments of the invention, a plant, subject,patient, organism or cell can be a non-human subject, patient, organismor cell. Thus, the invention provides a plant, animal or cell, producedby the present methods, or a progeny thereof. The progeny may be a cloneof the produced plant or animal, or may result from sexual reproductionby crossing with other individuals of the same species to introgressfurther desirable traits into their offspring. The cell may be in vivoor ex vivo in the cases of multicellular organisms, particularly animalsor plants. In the instance where the cell is in cultured, a cell linemay be established if appropriate culturing conditions are met andpreferably if the cell is suitably adapted for this purpose (forinstance a stem cell). Bacterial cell lines produced by the inventionare also envisaged. Hence, cell lines are also envisaged.

In some methods, the disease model can be used to study the effects ofmutations on the animal or cell and development and/or progression ofthe disease using measures commonly used in the study of the disease.Alternatively, such a disease model is useful for studying the effect ofa pharmaceutically active compound on the disease.

In some methods, the disease model can be used to assess the efficacy ofa potential gene therapy strategy. That is, a disease-associated gene orpolynucleotide can be modified such that the disease development and/orprogression is inhibited or reduced. In particular, the method comprisesmodifying a disease-associated gene or polynucleotide such that analtered protein is produced and, as a result, the animal or cell has analtered response. Accordingly, in some methods, a genetically modifiedanimal may be compared with an animal predisposed to development of thedisease such that the effect of the gene therapy event may be assessed.

In another embodiment, this invention provides a method of developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. The method comprises contacting a testcompound with a cell comprising one or more vectors that driveexpression of one or more of a CRISPR enzyme, and a direct repeatsequence linked to a guide sequence; and detecting a change in a readoutthat is indicative of a reduction or an augmentation of a cell signalingevent associated with, e.g., a mutation in a disease gene contained inthe cell.

A cell model or animal model can be constructed in combination with themethod of the invention for screening a cellular function change. Such amodel may be used to study the effects of a genome sequence modified bythe CRISPR complex of the invention on a cellular function of interest.For example, a cellular function model may be used to study the effectof a modified genome sequence on intracellular signaling orextracellular signaling. Alternatively, a cellular function model may beused to study the effects of a modified genome sequence on sensoryperception. In some such models, one or more genome sequences associatedwith a signaling biochemical pathway in the model are modified.

Several disease models have been specifically investigated. Theseinclude de novo autism risk genes CHD8, KATNAL2, and SCN2A; and thesyndromic autism (Angelman Syndrome) gene UBE3A. These genes andresulting autism models are of course preferred, but serve to show thebroad applicability of the invention across genes and correspondingmodels. An altered expression of one or more genome sequences associatedwith a signalling biochemical pathway can be determined by assaying fora difference in the mRNA levels of the corresponding genes between thetest model cell and a control cell, when they are contacted with acandidate agent. Alternatively, the differential expression of thesequences associated with a signaling biochemical pathway is determinedby detecting a difference in the level of the encoded polypeptide orgene product.

To assay for an agent-induced alteration in the level of mRNAtranscripts or corresponding polynucleotides, nucleic acid contained ina sample is first extracted according to standard methods in the art.For instance, mRNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook etal. (1989), or extracted by nucleic-acid-binding resins following theaccompanying instructions provided by the manufacturers. The mRNAcontained in the extracted nucleic acid sample is then detected byamplification procedures or conventional hybridization assays (e.g.Northern blot analysis) according to methods widely known in the art orbased on the methods exemplified herein.

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR. In particular, the isolated RNAcan be subjected to a reverse transcription assay that is coupled with aquantitative polymerase chain reaction (RT-PCR) in order to quantify theexpression level of a sequence associated with a signaling biochemicalpathway.

Detection of the gene expression level can be conducted in real time inan amplification assay. In one aspect, the amplified products can bedirectly visualized with fluorescent DNA-binding agents including butnot limited to DNA intercalators and DNA groove binders. Because theamount of the intercalators incorporated into the double-stranded DNAmolecules is typically proportional to the amount of the amplified DNAproducts, one can conveniently determine the amount of the amplifiedproducts by quantifying the fluorescence of the intercalated dye usingconventional optical systems in the art. DNA-binding dye suitable forthis application include SYBR green, SYBR blue, DAPI, propidium iodine,Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridineorange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, and the like.

In another aspect, other fluorescent labels such as sequence specificprobes can be employed in the amplification reaction to facilitate thedetection and quantification of the amplified products. Probe-basedquantitative amplification relies on the sequence-specific detection ofa desired amplified product. It utilizes fluorescent, target-specificprobes (e.g., TaqMan® probes) resulting in increased specificity andsensitivity. Methods for performing probe-based quantitativeamplification are well established in the art and are taught in U.S.Pat. No. 5,210,015.

In yet another aspect, conventional hybridization assays usinghybridization probes that share sequence homology with sequencesassociated with a signaling biochemical pathway can be performed.Typically, probes are allowed to form stable complexes with thesequences associated with a signaling biochemical pathway containedwithin the biological sample derived from the test subject in ahybridization reaction. It will be appreciated by one of skill in theart that where antisense is used as the probe nucleic acid, the targetpolynucleotides provided in the sample are chosen to be complementary tosequences of the antisense nucleic acids. Conversely, where thenucleotide probe is a sense nucleic acid, the target polynucleotide isselected to be complementary to sequences of the sense nucleic acid.

Hybridization can be performed under conditions of various stringency.Suitable hybridization conditions for the practice of the presentinvention are such that the recognition interaction between the probeand sequences associated with a signaling biochemical pathway is bothsufficiently specific and sufficiently stable. Conditions that increasethe stringency of a hybridization reaction are widely known andpublished in the art. See, for example, (Sambrook, et al., (1989);Nonradioactive In Situ Hybridization Application Manual, BoehringerMannheim, second edition). The hybridization assay can be formed usingprobes immobilized on any solid support, including but are not limitedto nitrocellulose, glass, silicon, and a variety of gene arrays. Apreferred hybridization assay is conducted on high-density gene chips asdescribed in U.S. Pat. No. 5,445,934.

For a convenient detection of the probe-target complexes formed duringthe hybridization assay, the nucleotide probes are conjugated to adetectable label. Detectable labels suitable for use in the presentinvention include any composition detectable by photochemical,biochemical, spectroscopic, immunochemical, electrical, optical orchemical means. A wide variety of appropriate detectable labels areknown in the art, which include fluorescent or chemiluminescent labels,radioactive isotope labels, enzymatic or other ligands. In preferredembodiments, one will likely desire to employ a fluorescent label or anenzyme tag, such as digoxigenin, ß-galactosidase, urease, alkalinephosphatase or peroxidase, avidin/biotin complex.

The detection methods used to detect or quantify the hybridizationintensity will typically depend upon the label selected above. Forexample, radiolabels may be detected using photographic film or aphosphoimager. Fluorescent markers may be detected and quantified usinga photodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and measuring thereaction product produced by the action of the enzyme on the substrate;and finally colorimetric labels are detected by simply visualizing thecolored label.

An agent-induced change in expression of sequences associated with asignalling biochemical pathway can also be determined by examining thecorresponding gene products. Determining the protein level typicallyinvolves a) contacting the protein contained in a biological sample withan agent that specifically bind to a protein associated with asignalling biochemical pathway; and (b) identifying any agent:proteincomplex so formed. In one aspect of this embodiment, the agent thatspecifically binds a protein associated with a signalling biochemicalpathway is an antibody, preferably a monoclonal antibody.

The reaction is performed by contacting the agent with a sample of theproteins associated with a signaling biochemical pathway derived fromthe test samples under conditions that will allow a complex to formbetween the agent and the proteins associated with a signallingbiochemical pathway. The formation of the complex can be detecteddirectly or indirectly according to standard procedures in the art. Inthe direct detection method, the agents are supplied with a detectablelabel and unreacted agents may be removed from the complex; the amountof remaining label thereby indicating the amount of complex formed. Forsuch method, it is preferable to select labels that remain attached tothe agents even during stringent washing conditions. It is preferablethat the label does not interfere with the binding reaction. In thealternative, an indirect detection procedure may use an agent thatcontains a label introduced either chemically or enzymatically. Adesirable label generally does not interfere with binding or thestability of the resulting agent:polypeptide complex. However, the labelis typically designed to be accessible to an antibody for an effectivebinding and hence generating a detectable signal.

A wide variety of labels suitable for detecting protein levels are knownin the art. Non-limiting examples include radioisotopes, enzymes,colloidal metals, fluorescent compounds, bioluminescent compounds, andchemiluminescent compounds.

The amount of agent:polypeptide complexes formed during the bindingreaction can be quantified by standard quantitative assays. Asillustrated above, the formation of agent:polypeptide complex can bemeasured directly by the amount of label remained at the site ofbinding. In an alternative, the protein associated with a signalingbiochemical pathway is tested for its ability to compete with a labeledanalog for binding sites on the specific agent. In this competitiveassay, the amount of label captured is inversely proportional to theamount of protein sequences associated with a signaling biochemicalpathway present in a test sample.

A number of techniques for protein analysis based on the generalprinciples outlined above are available in the art. They include but arenot limited to radioimmunoassays, ELISA (enzyme linked immunoradiometricassays), “sandwich” immunoassays, immunoradiometric assays, in situimmunoassays (using e.g., colloidal gold, enzyme or radioisotopelabels), western blot analysis, immunoprecipitation assays,immunofluorescent assays, and SDS-PAGE.

Antibodies that specifically recognize or bind to proteins associatedwith a signalling biochemical pathway are preferable for conducting theaforementioned protein analyses. Where desired, antibodies thatrecognize a specific type of post-translational modifications (e.g.,signaling biochemical pathway inducible modifications) can be used.Post-translational modifications include but are not limited toglycosylation, lipidation, acetylation, and phosphorylation. Theseantibodies may be purchased from commercial vendors. For example,anti-phosphotyrosine antibodies that specifically recognizetyrosine-phosphorylated proteins are available from a number of vendorsincluding Invitrogen and Perkin Elmer. Anti-phosphotyrosine antibodiesare particularly useful in detecting proteins that are differentiallyphosphorylated on their tyrosine residues in response to an ER stress.Such proteins include but are not limited to eukaryotic translationinitiation factor 2 alpha (eIF-2α). Alternatively, these antibodies canbe generated using conventional polyclonal or monoclonal antibodytechnologies by immunizing a host animal or an antibody-producing cellwith a target protein that exhibits the desired post-translationalmodification.

In practicing the subject method, it may be desirable to discern theexpression pattern of an protein associated with a signaling biochemicalpathway in different bodily tissue, in different cell types, and/or indifferent subcellular structures. These studies can be performed withthe use of tissue-specific, cell-specific or subcellular structurespecific antibodies capable of binding to protein markers that arepreferentially expressed in certain tissues, cell types, or subcellularstructures.

An altered expression of a gene associated with a signaling biochemicalpathway can also be determined by examining a change in activity of thegene product relative to a control cell. The assay for an agent-inducedchange in the activity of a protein associated with a signalingbiochemical pathway will dependent on the biological activity and/or thesignal transduction pathway that is under investigation. For example,where the protein is a kinase, a change in its ability to phosphorylatethe downstream substrate(s) can be determined by a variety of assaysknown in the art. Representative assays include but are not limited toimmunoblotting and immunoprecipitation with antibodies such asanti-phosphotyrosine antibodies that recognize phosphorylated proteins.In addition, kinase activity can be detected by high throughputchemiluminescent assays such as AlphaScreen™ (available from PerkinElmer) and eTag™ assay (Chan-Hui, et al. (2003) Clinical Immunology 111:162-174).

Where the protein associated with a signaling biochemical pathway ispart of a signaling cascade leading to a fluctuation of intracellular pHcondition, pH sensitive molecules such as fluorescent pH dyes can beused as the reporter molecules. In another example where the proteinassociated with a signaling biochemical pathway is an ion channel,fluctuations in membrane potential and/or intracellular ionconcentration can be monitored. A number of commercial kits andhigh-throughput devices are particularly suited for a rapid and robustscreening for modulators of ion channels. Representative instrumentsinclude FLIPR™ (Molecular Devices, Inc.) and VIPR (Aurora Biosciences).These instruments are capable of detecting reactions in over 1000 samplewells of a microplate simultaneously, and providing real-timemeasurement and functional data within a second or even a minisecond.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA).

Examples of target polynucleotides include a sequence associated with asignalling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif); that is, a short sequencerecognized by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are given inthe examples section below, and the skilled person will be able toidentify further PAM sequences for use with a given CRISPR enzyme.Further, engineering of the PAM Interacting (PI) domain may allowprograming of PAM specificity, improve target site recognition fidelity,and increase the versatility of the Cas, e.g. Cas9, genome engineeringplatform. Cas proteins, such as Cas9 proteins may be engineered to altertheir PAM specificity, for example as described in Kleinstiver B P etal. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.Nature. 2015 Jul. 23; 523(7561):481-5. doi: 10.1038/nature14592.

The target polynucleotide of a CRISPR complex may include a number ofdisease-associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes and polynucleotides as listed inU.S. provisional patent applications 61/736,527 and 61/748,427 havingBroad reference BI-2011/008/WSGR Docket No. 44063-701.101 andBI-2011/008/WSGR Docket No. 44063-701.102 respectively, both entitledSYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION filed on Dec.12, 2012 and Jan. 2, 2013, respectively, and PCT ApplicationPCT/US2013/074667, entitled DELIVERY, ENGINEERING AND OPTIMIZATION OFSYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION ANDTHERAPEUTIC APPLICATIONS, filed Dec. 12, 2013, the contents of all ofwhich are herein incorporated by reference in their entirety.

Examples of target polynucleotides include a sequence associated with asignalling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

Genome Wide Knock-Out Screening

The CRISPR proteins and systems described herein can be used to performefficient and cost effective functional genomic screens. Such screenscan utilize CRISPR effector protein based genome wide libraries. Suchscreens and libraries can provide for determining the function of genes,cellular pathways genes are involved in, and how any alteration in geneexpression can result in a particular biological process. An advantageof the present invention is that the CRISPR system avoids off-targetbinding and its resulting side effects. This is achieved using systemsarranged to have a high degree of sequence specificity for the targetDNA. In preferred embodiments of the invention, the CRISPR effectorprotein complexes are Cpf1 effector protein complexes.

In embodiments of the invention, a genome wide library may comprise aplurality of Cpf1guide RNAs, as described herein, comprising guidesequences that are capable of targeting a plurality of target sequencesin a plurality of genomic loci in a population of eukaryotic cells. Thepopulation of cells may be a population of embryonic stem (ES) cells.The target sequence in the genomic locus may be a non-coding sequence.The non-coding sequence may be an intron, regulatory sequence, splicesite, 3′ UTR, 5′ UTR, or polyadenylation signal. Gene function of one ormore gene products may be altered by said targeting. The targeting mayresult in a knockout of gene function. The targeting of a gene productmay comprise more than one guide RNA. A gene product may be targeted by2, 3, 4, 5, 6, 7, 8, 9, or 10 guide RNAs, preferably 3 to 4 per gene.Off-target modifications may be minimized by exploiting the staggereddouble strand breaks generated by Cpf1 effector protein complexes or byutilizing methods analogous to those used in CRISPR-Cas9 systems (See,e.g., DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V., Li,Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L A., Bao,G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013)),incorporated herein by reference. The targeting may be of about 100 ormore sequences. The targeting may be of about 1000 or more sequences.The targeting may be of about 20,000 or more sequences. The targetingmay be of the entire genome. The targeting may be of a panel of targetsequences focused on a relevant or desirable pathway. The pathway may bean immune pathway. The pathway may be a cell division pathway.

One aspect of the invention comprehends a genome wide library that maycomprise a plurality of Cpf1 guide RNAs that may comprise guidesequences that are capable of targeting a plurality of target sequencesin a plurality of genomic loci, wherein said targeting results in aknockout/knockdown of gene function. This library may potentiallycomprise guide RNAs that target each and every gene in the genome of anorganism.

In some embodiments of the invention the organism or subject is aeukaryote (including mammal including human) or a non-human eukaryote ora non-human animal or a non-human mammal. In some embodiments, theorganism or subject is a non-human animal, and may be an arthropod, forexample, an insect, or may be a nematode. In some methods of theinvention the organism or subject is a plant. In some methods of theinvention the organism or subject is a mammal or a non-human mammal. Anon-human mammal may be for example a rodent (preferably a mouse or arat), an ungulate, or a primate. In some methods of the invention theorganism or subject is algae, including microalgae, or is a fungus.

The knockout/knockdown of gene function may comprise: introducing intoeach cell in the population of cells a vector system of one or morevectors comprising an engineered, non-naturally occurring Cpf1 effectorprotein system comprising I. a Cpf1 effector protein, and II. one ormore guide RNAs, wherein components I and II may be same or on differentvectors of the system, integrating components I and II into each cell,wherein the guide sequence targets a unique gene in each cell, whereinthe Cpf1 effector protein is operably linked to a regulatory element,wherein when transcribed, the guide RNA comprising the guide sequencedirects sequence-specific binding of the Cpf1 effector protein system toa target sequence corresponding to the genomic loci of the unique gene,inducing cleavage of the genomic loci by the Cpf1 effector protein, andconfirming different knockout/knockdown mutations in a plurality ofunique genes in each cell of the population of cells thereby generatinga gene knockout/knockdown cell library. The invention comprehends thatthe population of cells is a population of eukaryotic cells, and in apreferred embodiment, the population of cells is a population ofembryonic stem (ES) cells.

The one or more vectors may be plasmid vectors. The vector may be asingle vector comprising a Cpf1 effector protein, a gRNA, andoptionally, a selection marker into target cells. Not being bound by atheory, the ability to simultaneously deliver a Cpf1 effector proteinand gRNA through a single vector enables application to any cell type ofinterest, without the need to first generate cell lines that express theCpf1 effector protein. The regulatory element may be an induciblepromoter. The inducible promoter may be a doxycycline induciblepromoter. In some methods of the invention the expression of the guidesequence is under the control of the T7 promoter and is driven by theexpression of T7 polymerase. The confirming of differentknockout/knockdown mutations may be by whole exome sequencing. Theknockout/knockdown mutation may be achieved in 100 or more unique genes.The knockout/knockdown mutation may be achieved in 1000 or more uniquegenes. The knockout/knockdown mutation may be achieved in 20,000 or moreunique genes. The knockout/knockdown mutation may be achieved in theentire genome. The knockout/knockdown of gene function may be achievedin a plurality of unique genes which function in a particularphysiological pathway or condition. The pathway or condition may be animmune pathway or condition. The pathway or condition may be a celldivision pathway or condition.

The invention also provides kits that comprise the genome wide librariesmentioned herein. The kit may comprise a single container comprisingvectors or plasmids comprising the library of the invention. The kit mayalso comprise a panel comprising a selection of unique Cpf1 effectorprotein system guide RNAs comprising guide sequences from the library ofthe invention, wherein the selection is indicative of a particularphysiological condition. The invention comprehends that the targeting isof about 100 or more sequences, about 1000 or more sequences or about20,000 or more sequences or the entire genome. Furthermore, a panel oftarget sequences may be focused on a relevant or desirable pathway, suchas an immune pathway or cell division.

In an additional aspect of the invention, the Cpf1 effector protein maycomprise one or more mutations and may be used as a generic DNA bindingprotein with or without fusion to a functional domain. The mutations maybe artificially introduced mutations or gain- or loss-of-functionmutations. The mutations have been characterized as described herein. Inone aspect of the invention, the functional domain may be atranscriptional activation domain, which may be VP64. In other aspectsof the invention, the functional domain may be a transcriptionalrepressor domain, which may be KRAB or SID4X. Other aspects of theinvention relate to the mutated Cpf1 effector protein being fused todomains which include but are not limited to a transcriptionalactivator, repressor, a recombinase, a transposase, a histone remodeler,a demethylase, a DNA methyltransferase, a cryptochrome, a lightinducible/controllable domain or a chemically inducible/controllabledomain. Some methods of the invention can include inducing expression oftargeted genes. In one embodiment, inducing expression by targeting aplurality of target sequences in a plurality of genomic loci in apopulation of eukaryotic cells is by use of a functional domain.

Useful in the practice of the instant invention utilizing Cpf1 effectorprotein complexes are methods used in CRISPR-Cas9 systems and referenceis made to:

Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O.,Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T.,Heckl, D., Ebert, BL., Root, D E., Doench, J G., Zhang, F. ScienceDecember 12. (2013). [Epub ahead of print]; Published in final editedform as: Science. 2014 Jan. 3; 343(6166): 84-87.

Shalem et al. involves a new way to interrogate gene function on agenome-wide scale. Their studies showed that delivery of a genome-scaleCRISPR-Cas9 knockout (GeCKO) library targeted 18,080 genes with 64,751unique guide sequences enabled both negative and positive selectionscreening in human cells. First, the authors showed use of the GeCKOlibrary to identify genes essential for cell viability in cancer andpluripotent stem cells. Next, in a melanoma model, the authors screenedfor genes whose loss is involved in resistance to vemurafenib, atherapeutic that inhibits mutant protein kinase BRAF. Their studiesshowed that the highest-ranking candidates included previously validatedgenes NF1 and MED12 as well as novel hitsNF2, CUL3, TADA2B, and TADA1.The authors observed a high level of consistency between independentguide RNAs targeting the same gene and a high rate of hit confirmation,and thus demonstrated the promise of genome-scale screening with Cas9.

Reference is also made to US patent publication number US20140357530;and PCT Patent Publication WO2014093701, hereby incorporated herein byreference. Reference is also made to NIH Press Release of Oct. 22, 2015entitled, “Researchers identify potential alternative to CRISPR-Casgenome editing tools: New Cas enzymes shed light on evolution ofCRISPR-Cas systems, which is incorporated by reference.

Functional Alteration and Screening

In another aspect, the present invention provides for a method offunctional evaluation and screening of genes. The use of the CRISPRsystem of the present invention to precisely deliver functional domains,to activate or repress genes or to alter epigenetic state by preciselyaltering the methylation site on a specific locus of interest, can bewith one or more guide RNAs applied to a single cell or population ofcells or with a library applied to genome in a pool of cells ex vivo orin vivo comprising the administration or expression of a librarycomprising a plurality of guide RNAs (gRNAs) and wherein the screeningfurther comprises use of a Cpf1 effector protein, wherein the CRISPRcomplex comprising the Cpf1 effector protein is modified to comprise aheterologous functional domain. In an aspect the invention provides amethod for screening a genome comprising the administration to a host orexpression in a host in vivo of a library. In an aspect the inventionprovides a method as herein discussed further comprising an activatoradministered to the host or expressed in the host. In an aspect theinvention provides a method as herein discussed wherein the activator isattached to a Cpf1 effector protein. In an aspect the invention providesa method as herein discussed wherein the activator is attached to the Nterminus or the C terminus of the Cpf1 effector protein. In an aspectthe invention provides a method as herein discussed wherein theactivator is attached to a gRNA loop. In an aspect the inventionprovides a method as herein discussed further comprising a repressoradministered to the host or expressed in the host. In an aspect theinvention provides a method as herein discussed, wherein the screeningcomprises affecting and detecting gene activation, gene inhibition, orcleavage in the locus.

In an aspect, the invention provides efficient on-target activity andminimizes off target activity. In an aspect, the invention providesefficient on-target cleavage by Cpf1 effector protein and minimizesoff-target cleavage by the Cpf1 effector protein. In an aspect, theinvention provides guide specific binding of Cpf1 effector protein at agene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides target-specific gene regulation. In an aspect, theinvention provides guide specific binding of Cpf1 effector protein at agene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides for cleavage at one gene locus and gene regulation ata different gene locus using a single Cpf1 effector protein. In anaspect, the invention provides orthogonal activation and/or inhibitionand/or cleavage of multiple targets using one or more Cpf1 effectorprotein and/or enzyme.

In an aspect the invention provides a method as herein discussed,wherein the host is a eukaryotic cell. In an aspect the inventionprovides a method as herein discussed, wherein the host is a mammaliancell. In an aspect the invention provides a method as herein discussed,wherein the host is a non-human eukaryote. In an aspect the inventionprovides a method as herein discussed, wherein the non-human eukaryoteis a non-human mammal. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal is a mouse. An aspect theinvention provides a method as herein discussed comprising the deliveryof the Cpf1 effector protein complexes or component(s) thereof ornucleic acid molecule(s) coding therefor, wherein said nucleic acidmolecule(s) are operatively linked to regulatory sequence(s) andexpressed in vivo. In an aspect the invention provides a method asherein discussed wherein the expressing in vivo is via a lentivirus, anadenovirus, or an AAV. In an aspect the invention provides a method asherein discussed wherein the delivery is via a particle, a nanoparticle,a lipid or a cell penetrating peptide (CPP).

In an aspect the invention provides a pair of CRISPR complexescomprising Cpf1 effector protein, each comprising a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein at least one loop ofeach gRNA is modified by the insertion of distinct RNA sequence(s) thatbind to one or more adaptor proteins, and wherein the adaptor protein isassociated with one or more functional domains, wherein each gRNA ofeach Cpf1 effector protein complex comprises a functional domain havinga DNA cleavage activity. In an aspect the invention provides paired Cpf1effector protein complexes as herein-discussed, wherein the DNA cleavageactivity is due to a Fok1 nuclease.

In an aspect the invention provides a method for cutting a targetsequence in a genomic locus of interest comprising delivery to a cell ofthe Cpf1 effector protein complexes or component(s) thereof or nucleicacid molecule(s) coding therefor, wherein said nucleic acid molecule(s)are operatively linked to regulatory sequence(s) and expressed in vivo.In an aspect the invention provides a method as herein-discussed whereinthe delivery is via a lentivirus, an adenovirus, or an AAV. In an aspectthe invention provides a method as herein-discussed or paired Cpf1effector protein complexes as herein-discussed wherein the targetsequence for a first complex of the pair is on a first strand of doublestranded DNA and the target sequence for a second complex of the pair ison a second strand of double stranded DNA. In an aspect the inventionprovides a method as herein-discussed or paired Cpf1 effector proteincomplexes as herein-discussed wherein the target sequences of the firstand second complexes are in proximity to each other such that the DNA iscut in a manner that facilitates homology directed repair. In an aspecta herein method can further include introducing into the cell templateDNA. In an aspect a herein method or herein paired Cpf1 effector proteincomplexes can involve wherein each Cpf1 effector protein complex has aCpf1 effector enzyme that is mutated such that it has no more than about5% of the nuclease activity of the Cpf1 effector enzyme that is notmutated.

In an aspect the invention provides a library, method or complex asherein-discussed wherein the gRNA is modified to have at least onenon-coding functional loop, e.g., wherein the at least one non-codingfunctional loop is repressive; for instance, wherein the at least onenon-coding functional loop comprises Alu.

In one aspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring CRISPR system comprisinga Cpf1 effector protein and guide RNA that targets the DNA molecule,whereby the guide RNA targets the DNA molecule encoding the gene productand the Cpf1 effector protein cleaves the DNA molecule encoding the geneproduct, whereby expression of the gene product is altered; and, whereinthe Cpf1 effector protein and the guide RNA do not naturally occurtogether. The invention comprehends the guide RNA comprising a guidesequence linked to a direct repeat sequence. The invention furthercomprehends the Cpf1 effector protein being codon optimized forexpression in a Eukaryotic cell. In a preferred embodiment theEukaryotic cell is a mammalian cell and in a more preferred embodimentthe mammalian cell is a human cell. In a further embodiment of theinvention, the expression of the gene product is decreased.

In some embodiments, one or more functional domains are associated withthe Cpf1 effector protein. In some embodiments, one or more functionaldomains are associated with an adaptor protein, for example as used withthe modified guides of Konnerman et al. (Nature 517, 583-588, 29 Jan.2015). In some embodiments, one or more functional domains areassociated with an dead gRNA (dRNA). In some embodiments, a dRNA complexwith active Cpf1 effector protein directs gene regulation by afunctional domain at on gene locus while an gRNA directs DNA cleavage bythe active Cpf1 effector protein at another locus, for example asdescribed analogously in CRISPR-Cas9 systems by Dahlman et al.,‘Orthogonal gene control with a catalytically active Cas9 nuclease’ (inpress). In some embodiments, dRNAs are selected to maximize selectivityof regulation for a gene locus of interest compared to off-targetregulation. In some embodiments, dRNAs are selected to maximize targetgene regulation and minimize target cleavage

For the purposes of the following discussion, reference to a functionaldomain could be a functional domain associated with the Cpf1 effectorprotein or a functional domain associated with the adaptor protein.

In the practice of the invention, loops of the gRNA may be extended,without colliding with the Cpf1 protein by the insertion of distinct RNAloop(s) or distinct sequence(s) that may recruit adaptor proteins thatcan bind to the distinct RNA loop(s) or distinct sequence(s). Theadaptor proteins may include but are not limited to orthogonalRNA-binding protein/aptamer combinations that exist within the diversityof bacteriophage coat proteins. A list of such coat proteins includes,but is not limited to: Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34,JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5,ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. These adaptor proteins or orthogonalRNA binding proteins can further recruit effector proteins or fusionswhich comprise one or more functional domains. In some embodiments, thefunctional domain may be selected from the group consisting of:transposase domain, integrase domain, recombinase domain, resolvasedomain, invertase domain, protease domain, DNA methyltransferase domain,DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylasedomain, histone deacetylases domain, nuclease domain, repressor domain,activator domain, nuclear-localization signal domains,transcription-regulatory protein (or transcription complex recruiting)domain, cellular uptake activity associated domain, nucleic acid bindingdomain, antibody presentation domain, histone modifying enzymes,recruiter of histone modifying enzymes; inhibitor of histone modifyingenzymes, histone methyltransferase, histone demethylase, histone kinase,histone phosphatase, histone ribosylase, histone deribosylase, histoneubiquitinase, histone deubiquitinase, histone biotinase and histone tailprotease. In some preferred embodiments, the functional domain is atranscriptional activation domain, such as, without limitation, VP64,p65, MyoD1, HSF1, RTA, SETT/9 or a histone acetyltransferase. In someembodiments, the functional domain is a transcription repression domain,preferably KRAB. In some embodiments, the transcription repressiondomain is SID, or concatemers of SID (eg SID4X). In some embodiments,the functional domain is an epigenetic modifying domain, such that anepigenetic modifying enzyme is provided. In some embodiments, thefunctional domain is an activation domain, which may be the P65activation domain.

In some embodiments, the one or more functional domains is an NLS(Nuclear Localization Sequence) or an NES (Nuclear Export Signal). Insome embodiments, the one or more functional domains is atranscriptional activation domain comprises VP64, p65, MyoD1, HSF1, RTA,SET7/9 and a histone acetyltransferase. Other references herein toactivation (or activator) domains in respect of those associated withthe CRISPR enzyme include any known transcriptional activation domainand specifically VP64, p65, MyoD1, HSF1, RTA, SET7/9 or a histoneacetyltransferase.

In some embodiments, the one or more functional domains is atranscriptional repressor domain. In some embodiments, thetranscriptional repressor domain is a KRAB domain. In some embodiments,the transcriptional repressor domain is a NuE domain, NcoR domain, SIDdomain or a SID4X domain.

In some embodiments, the one or more functional domains have one or moreactivities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,RNA cleavage activity, DNA cleavage activity, DNA integration activityor nucleic acid binding activity.

Histone modifying domains are also preferred in some embodiments.Exemplary histone modifying domains are discussed below. Transposasedomains, HR (Homologous Recombination) machinery domains, recombinasedomains, and/or integrase domains are also preferred as the presentfunctional domains. In some embodiments, DNA integration activityincludes HR machinery domains, integrase domains, recombinase domainsand/or transposase domains. Histone acetyltransferases are preferred insome embodiments.

In some embodiments, the DNA cleavage activity is due to a nuclease. Insome embodiments, the nuclease comprises a Fok1 nuclease. See, “DimericCRISPR RNA-guided FokI nucleases for highly specific genome editing”,Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden,Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J.Keith Joung Nature Biotechnology 32(6): 569-77 (2014), relates todimeric RNA-guided FokI Nucleases that recognize extended sequences andcan edit endogenous genes with high efficiencies in human cells.

In some embodiments, the one or more functional domains is attached tothe Cpf1 effector protein so that upon binding to the sgRNA and targetthe functional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function.

In some embodiments, the one or more functional domains is attached tothe adaptor protein so that upon binding of the Cpf1 effector protein tothe gRNA and target, the functional domain is in a spatial orientationallowing for the functional domain to function in its attributedfunction.

In an aspect the invention provides a composition as herein discussedwherein the one or more functional domains is attached to the Cpf1effector protein or adaptor protein via a linker, optionally a GlySerlinker, as discussed herein.

Endogenous transcriptional repression is often mediated by chromatinmodifying enzymes such as histone methyltransferases (HMTs) anddeacetylases (HDACs). Repressive histone effector domains are known andan exemplary list is provided below. In the exemplary table, preferencewas given to proteins and functional truncations of small size tofacilitate efficient viral packaging (for instance via AAV). In general,however, the domains may include HDACs, histone methyltransferases(HMTs), and histone acetyltransferase (HAT) inhibitors, as well as HDACand HMT recruiting proteins. The functional domain may be or include, insome embodiments, HDAC Effector Domains, HDAC Recruiter EffectorDomains, Histone Methyltransferase (HMT) Effector Domains, HistoneMethyltransferase (HMT) Recruiter Effector Domains, or HistoneAcetyltransferase Inhibitor Effector Domains.

HDAC Effector Domains Subtype/ Substrate Modification Full SelectedFinal Catalytic Complex Name (if known) (if known) Organism size (aa)truncation (aa) size (aa) domain HDAC I HDAC8 — — X. laevis 325 1-325325 1-272: HDAC HDAC I RPD3 — — S. cerevisiae 433 19-340  322 19-331:(Vannier) HDAC HDAC MesoLo4 — — M. loti 300 1-300 300 — IV (Gregoretti)HDAC HDAC11 — — H. sapiens 347 1-347 (Gao) 347 14-326: IV HDAC HD2 HDT1— — A. thaliana 245 1-211 (Wu) 211 — SIRT I SIRT3 H3K9Ac — H. sapiens399 143-399 257 126-382: H4K16Ac (Scher) SIRT H3K56Ac SIRT I HST2 — — C.albicans 331 1-331 (Hnisz) 331 — SIRT I CobB — — E. coli (K12) 242 1-242(Landry) 242 — SIRT I HST2 — — S. cerevisiae 357 8-298 (Wilson) 291 —SIRT III SIRT5 H4K8Ac — H. sapiens 310 37-310 (Gertz) 274 41-309:H4K16Ac SIRT SIRT III Sir2A — — P. falciparum 273 1-273 (Zhu) 27319-273: SIRT SIRT IV SIRT6 H3K9Ac — H. sapiens 355 1-289 289 35-274:H3K56Ac (Tennen) SIRT

Accordingly, the repressor domains of the present invention may beselected from histone methyltransferases (HMTs), histone deacetylases(HDACs), histone acetyltransferase (HAT) inhibitors, as well as HDAC andHMT recruiting proteins.

The HDAC domain may be any of those in the table above, namely: HDAC8,RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB, HST2, SIRT5, Sir2A, orSIRT6.

In some embodiment, the functional domain may be a HDAC RecruiterEffector Domain. Preferred examples include those in the Table below,namely MeCP2, MBD2b, Sin3a, NcoR, SALL1, RCOR1. NcoR is exemplified inthe present Examples and, although preferred, it is envisaged thatothers in the class will also be useful.

Table of HDAC Recruiter Effector Domains Subtype/ Substrate ModificationFull Selected Final Catalytic Complex Name (if known) (if known)Organism size (aa) truncation (aa) size (aa) domain Sin3a MeCP2 — — R.norvegicus 492 207-492 (Nan) 286 — Sin3a MBD2b — — H. sapiens 262 45-262(Boeke) 218 — Sin3a Sin3a — — H. sapiens 1273 524-851 328 627-829: HDAC1(Laherty) interaction NcoR NcoR — — H. sapiens 2440 420-488 69 — (Zhang)NuRD SALL1 — — M. musculus 1322 1-93 (Lauberth) 93 — CoREST RCOR1 — — H.sapiens 482 81-300 (Gu, 220 — Ouyang)

In some embodiment, the functional domain may be a Methyltransferase(HMT) Effector Domain. Preferred examples include those in the Tablebelow, namely NUE, vSET, EHMT2/G9A, SUV39H1, dim-5, KYP, SUVR4, SET4,SET1, SETD8, and TgSET8. NUE is exemplified in the present Examples and,although preferred, it is envisaged that others in the class will alsobe useful.

Table of Histone Methyltransferase (HMT) Effector Domains Subtype/Substrate Modification Full Selected Final Catalytic Complex Name (ifknown) (if known) Organism size (aa) truncation (aa) size (aa) domainSET NUE H2B, — C. trachomatis 219 1-219 219 — H3, H4 (Pennini) SET vSET— H3K27me3 P. bursaria 119 1-119 119 4-112: chlorella (Mujtaba) SET2virus SUV39 EHMT2/ H1.4K2, H3K9me1/2, M. musculus 1263 969-1263 2951025-1233: family G9A H3K9, H1K25me1 (Tachibana) preSET, H3K27 SET,postSET SUV39 SUV39H1 — H3K9me2/3 H. sapiens 412 79-412 334 172-412:(Snowden) preSET, SET, postSET Suvar3-9 dim-5 — H3K9me3 N. crassa 3311-331 331 77-331: (Rathert) preSET, SET, postSET Suvar3-9 KYP —H3K9me1/2 A. thaliana 624 335-601 267 — (SUVH (Jackson) subfamily)Suvar3-9 SUVR4 H3K9me1 H3K9me2/3 A. thaliana 492 180-492 313 192-462:(SUVR (Thorstensen) preSET, subfamily) SET, postSET Suvar4-20 SET4 —H4K20me3 C. elegans 288 1-288 (Vielle) 288 — SET8 SET1 — H4K20me1 C.elegans 242 1-242 (Vielle) 242 — SET8 SETD8 — H4K20me1 H. sapiens 393185-393 209 256-382: (Couture) SET SET8 TgSET8 — H4K20me1/ T. gondii1893 1590-1893 304 1749-1884: 2/3 (Sautel) SET

In some embodiment, the functional domain may be a HistoneMethyltransferase (HMT) Recruiter Effector Domain. Preferred examplesinclude those in the Table below, namely Hp1a, PHF19, and NIPP1.

Table of Histone Methyltransferase (HMT) Recruiter Effector DomainsSubtype/ Substrate Modification Full Selected Final Catalytic ComplexName (if known) (if known) Organism size (aa) truncation (aa) size (aa)domain — Hp1a — H3K9me3 M. musculus 191 73-191 119 121-179: (Hathaway)chromoshadow — PHF19 — H3K27me3 H. sapiens 580 (1-250) + GGSG 335163-250: PHD2 linker + (Ballaré) (500-580) — NIPP1 — H3K27me3 H. sapiens351 1-329 (Jin) 329 310-329: EED

In some embodiment, the functional domain may be HistoneAcetyltransferase Inhibitor Effector Domain. Preferred examples includeSET/TAF-1β listed in the Table below.

Table of Histone Acetyltransferase Inhibitor Effector Domains Subtype/Substrate Modification Full Selected Final Catalytic Complex Name (ifknown) (if known) Organism size (aa) truncation (aa) size (aa) domain —SET/TAF-1β — — M. musculus 289 1-289 289 — (Cervoni)

It is also preferred to target endogenous (regulatory) control elements(such as enhancers and silencers) in addition to a promoter orpromoter-proximal elements. Thus, the invention can also be used totarget endogenous control elements (including enhancers and silencers)in addition to targeting of the promoter. These control elements can belocated upstream and downstream of the transcriptional start site (TSS),starting from 200 bp from the TSS to 100 kb away. Targeting of knowncontrol elements can be used to activate or repress the gene ofinterest. In some cases, a single control element can influence thetranscription of multiple target genes. Targeting of a single controlelement could therefore be used to control the transcription of multiplegenes simultaneously.

Targeting of putative control elements on the other hand (e.g. by tilingthe region of the putative control element as well as 200 bp up to 100kB around the element) can be used as a means to verify such elements(by measuring the transcription of the gene of interest) or to detectnovel control elements (e.g. by tiling 100 kb upstream and downstream ofthe TSS of the gene of interest). In addition, targeting of putativecontrol elements can be useful in the context of understanding geneticcauses of disease. Many mutations and common SNP variants associatedwith disease phenotypes are located outside coding regions. Targeting ofsuch regions with either the activation or repression systems describedherein can be followed by readout of transcription of either a) a set ofputative targets (e.g. a set of genes located in closest proximity tothe control element) or b) whole-transcriptome readout by e.g. RNAseq ormicroarray. This would allow for the identification of likely candidategenes involved in the disease phenotype. Such candidate genes could beuseful as novel drug targets.

Histone acetyltransferase (HAT) inhibitors are mentioned herein.However, an alternative in some embodiments is for the one or morefunctional domains to comprise an acetyltransferase, preferably ahistone acetyltransferase. These are useful in the field of epigenomics,for example in methods of interrogating the epigenome. Methods ofinterrogating the epigenome may include, for example, targetingepigenomic sequences. Targeting epigenomic sequences may include theguide being directed to an epigenomic target sequence. Epigenomic targetsequence may include, in some embodiments, include a promoter, silenceror an enhancer sequence.

Use of a functional domain linked to a Cpf1 effector protein asdescribed herein, preferably a dead-Cpf1 effector protein, morepreferably a dead-FnCpf1 effector protein, to target epigenomicsequences can be used to activate or repress promoters, silencer orenhancers.

Examples of acetyltransferases are known but may include, in someembodiments, histone acetyltransferases. In some embodiments, thehistone acetyltransferase may comprise the catalytic core of the humanacetyltransferase p300 (Gerbasch & Reddy, Nature Biotech 6 Apr. 2015).

In some preferred embodiments, the functional domain is linked to adead-Cpf1 effector protein to target and activate epigenomic sequencessuch as promoters or enhancers. One or more guides directed to suchpromoters or enhancers may also be provided to direct the binding of theCRISPR enzyme to such promoters or enhancers.

The term “associated with” is used here in relation to the associationof the functional domain to the Cpf1 effector protein or the adaptorprotein. It is used in respect of how one molecule ‘associates’ withrespect to another, for example between an adaptor protein and afunctional domain, or between the Cpf1 effector protein and a functionaldomain. In the case of such protein-protein interactions, thisassociation may be viewed in terms of recognition in the way an antibodyrecognizes an epitope. Alternatively, one protein may be associated withanother protein via a fusion of the two, for instance one subunit beingfused to another subunit. Fusion typically occurs by addition of theamino acid sequence of one to that of the other, for instance viasplicing together of the nucleotide sequences that encode each proteinor subunit. Alternatively, this may essentially be viewed as bindingbetween two molecules or direct linkage, such as a fusion protein. Inany event, the fusion protein may include a linker between the twosubunits of interest (i.e. between the enzyme and the functional domainor between the adaptor protein and the functional domain). Thus, in someembodiments, the Cpf1 effector protein or adaptor protein is associatedwith a functional domain by binding thereto. In other embodiments, theCpf1 effector protein or adaptor protein is associated with a functionaldomain because the two are fused together, optionally via anintermediate linker.

Attachment of a functional domain or fusion protein can be via a linker,e.g., a flexible glycine-serine (GlyGlyGlySer) or (GGGS)₃ or a rigidalpha-helical linker such as (Ala(GluAlaAlaAlaLys)Ala). Linkers such as(GGGGS)₃ are preferably used herein to separate protein or peptidedomains. (GGGGS)₃ is preferable because it is a relatively long linker(15 amino acids). The glycine residues are the most flexible and theserine residues enhance the chance that the linker is on the outside ofthe protein. (GGGGS)₆ (GGGGS)₉ or (GGGGS)₁₂ may preferably be used asalternatives. Other preferred alternatives are (GGGGS)₁, (GGGGS)₂,(GGGGS)₄, (GGGGS)₅, (GGGGS)₇, (GGGGS)₈, (GGGGS)₁₀, or (GGGGS)₁₁.Alternative linkers are available, but highly flexible linkers arethought to work best to allow for maximum opportunity for the 2 parts ofthe Cpf1 to come together and thus reconstitute Cpf1 activity. Onealternative is that the NLS of nucleoplasmin can be used as a linker.For example, a linker can also be used between the Cpf1 and anyfunctional domain. Again, a (GGGGS)₃ linker may be used here (or the 6,9, or 12 repeat versions therefore) or the NLS of nucleoplasmin can beused as a linker between Cpf1 and the functional domain.

Saturating Mutagenesis

The Cpf1 effector protein system(s) described herein can be used toperform saturating or deep scanning mutagenesis of genomic loci inconjunction with a cellular phenotype—for instance, for determiningcritical minimal features and discrete vulnerabilities of functionalelements required for gene expression, drug resistance, and reversal ofdisease. By saturating or deep scanning mutagenesis is meant that everyor essentially every DNA base is cut within the genomic loci. A libraryof Cpf1 effector protein guide RNAs may be introduced into a populationof cells. The library may be introduced, such that each cell receives asingle guide RNA (gRNA). In the case where the library is introduced bytransduction of a viral vector, as described herein, a low multiplicityof infection (MOI) is used. The library may include gRNAs targetingevery sequence upstream of a (protospacer adjacent motif) (PAM) sequencein a genomic locus. The library may include at least 100 non-overlappinggenomic sequences upstream of a PAM sequence for every 1000 base pairswithin the genomic locus. The library may include gRNAs targetingsequences upstream of at least one different PAM sequence. The Cpf1effector protein systems may include more than one Cpf1 protein. AnyCpf1 effector protein as described herein, including orthologues orengineered Cpf1 effector proteins that recognize different PAM sequencesmay be used. The frequency of off target sites for a gRNA may be lessthan 500. Off target scores may be generated to select gRNAs with thelowest off target sites. Any phenotype determined to be associated withcutting at a gRNA target site may be confirmed by using gRNAs targetingthe same site in a single experiment. Validation of a target site mayalso be performed by using a modified Cpf1 effector protein, asdescribed herein, and two gRNAs targeting the genomic site of interest.Not being bound by a theory, a target site is a true hit if the changein phenotype is observed in validation experiments.

The genomic loci may include at least one continuous genomic region. Theat least one continuous genomic region may comprise up to the entiregenome. The at least one continuous genomic region may comprise afunctional element of the genome. The functional element may be within anon-coding region, coding gene, intronic region, promoter, or enhancer.The at least one continuous genomic region may comprise at least 1 kb,preferably at least 50 kb of genomic DNA. The at least one continuousgenomic region may comprise a transcription factor binding site. The atleast one continuous genomic region may comprise a region of DNase Ihypersensitivity. The at least one continuous genomic region maycomprise a transcription enhancer or repressor element. The at least onecontinuous genomic region may comprise a site enriched for an epigeneticsignature. The at least one continuous genomic DNA region may comprisean epigenetic insulator. The at least one continuous genomic region maycomprise two or more continuous genomic regions that physicallyinteract. Genomic regions that interact may be determined by ‘4Ctechnology’. 4C technology allows the screening of the entire genome inan unbiased manner for DNA segments that physically interact with a DNAfragment of choice, as is described in Zhao et al. ((2006) Nat Genet 38,1341-7) and in U.S. Pat. No. 8,642,295, both incorporated herein byreference in its entirety. The epigenetic signature may be histoneacetylation, histone methylation, histone ubiquitination, histonephosphorylation, DNA methylation, or a lack thereof.

The Cpf1 effector protein system(s) for saturating or deep scanningmutagenesis can be used in a population of cells. The Cpf1 effectorprotein system(s) can be used in eukaryotic cells, including but notlimited to mammalian and plant cells. The population of cells may beprokaryotic cells. The population of eukaryotic cells may be apopulation of embryonic stem (ES) cells, neuronal cells, epithelialcells, immune cells, endocrine cells, muscle cells, erythrocytes,lymphocytes, plant cells, or yeast cells.

In one aspect, the present invention provides for a method of screeningfor functional elements associated with a change in a phenotype. Thelibrary may be introduced into a population of cells that are adapted tocontain a Cpf1 effector protein. The cells may be sorted into at leasttwo groups based on the phenotype. The phenotype may be expression of agene, cell growth, or cell viability. The relative representation of theguide RNAs present in each group are determined, whereby genomic sitesassociated with the change in phenotype are determined by therepresentation of guide RNAs present in each group. The change inphenotype may be a change in expression of a gene of interest. The geneof interest may be upregulated, downregulated, or knocked out. The cellsmay be sorted into a high expression group and a low expression group.The population of cells may include a reporter construct that is used todetermine the phenotype. The reporter construct may include a detectablemarker. Cells may be sorted by use of the detectable marker.

In another aspect, the present invention provides for a method ofscreening for genomic sites associated with resistance to a chemicalcompound. The chemical compound may be a drug or pesticide. The librarymay be introduced into a population of cells that are adapted to containa Cpf1 effector protein, wherein each cell of the population contains nomore than one guide RNA; the population of cells are treated with thechemical compound; and the representation of guide RNAs are determinedafter treatment with the chemical compound at a later time point ascompared to an early time point, whereby genomic sites associated withresistance to the chemical compound are determined by enrichment ofguide RNAs. Representation of gRNAs may be determined by deep sequencingmethods.

Useful in the practice of the instant invention utilizing Cpf1 effectorprotein complexes are methods used in CRISPR-Cas9 systems and referenceis made to the article entitled BCL11A enhancer dissection byCas9-mediated in situ saturating mutagenesis. Canver, M. C., Smith, E.C., Sher, F., Pinello, L., Sanjana, N. E., Shalem, O., Chen, D. D.,Schupp, P. G., Vinjamur, D. S., Garcia, S. P., Luc, S., Kurita, R.,Nakamura, Y., Fujiwara, Y., Maeda, T., Yuan, G., Zhang, F., Orkin, S.H., & Bauer, D. E. DOI:10.1038/nature15521, published online Sep. 16,2015, the article is herein incorporated by reference and discussedbriefly below:

Canver et al. involves novel pooled CRISPR-Cas9 guide RNA libraries toperform in situ saturating mutagenesis of the human and mouse BCL11Aerythroid enhancers previously identified as an enhancer associated withfetal hemoglobin (HbF) level and whose mouse ortholog is necessary forerythroid BCL11A expression. This approach revealed critical minimalfeatures and discrete vulnerabilities of these enhancers. Throughediting of primary human progenitors and mouse transgenesis, the authorsvalidated the BCL11A erythroid enhancer as a target for HbF reinduction.The authors generated a detailed enhancer map that informs therapeuticgenome editing.

Method of Using Cpf1 Systems to Modify a Cell or Organism

The invention in some embodiments comprehends a method of modifying ancell or organism. The cell may be a prokaryotic cell or a eukaryoticcell. The cell may be a mammalian cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Thecell may also be a plant cell. The plant cell may be of a crop plantsuch as cassava, corn, sorghum, wheat, or rice. The plant cell may alsobe of an algae, tree or vegetable. The modification introduced to thecell by the present invention may be such that the cell and progeny ofthe cell are altered for improved production of biologic products suchas an antibody, starch, alcohol or other desired cellular output. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell include an alteration that changesthe biologic product produced.

The system may comprise one or more different vectors. In an aspect ofthe invention, the Cas protein is codon optimized for expression thedesired cell type, preferentially a eukaryotic cell, preferably amammalian cell or a human cell.

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and ψ2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

Delivery

The invention involves at least one component of the CRISPR complex,e.g., RNA, delivered via at least one nanoparticle complex. In someaspects, the invention provides methods comprising delivering one ormore polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and animals comprisingor produced from such cells. In some embodiments, a CRISPR enzyme incombination with (and optionally complexed with) a guide sequence isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a CRISPR system to cells in culture, or ina host organism. Non-viral vector delivery systems include DNA plasmids,RNA (e.g. a transcript of a vector described herein), naked nucleicacid, and nucleic acid complexed with a delivery vehicle, such as aliposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. For a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Bohm (eds) (1995); and Yu et al., GeneTherapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described ine.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) andlipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In another embodiment, Cocal vesiculovirus envelope pseudotypedretroviral vector particles are contemplated (see, e.g., US PatentPublication No. 20120164118 assigned to the Fred Hutchinson CancerResearch Center). Cocal virus is in the Vesiculovirus genus, and is acausative agent of vesicular stomatitis in mammals. Cocal virus wasoriginally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet.Res. 25:236-242 (1964)), and infections have been identified inTrinidad, Brazil, and Argentina from insects, cattle, and horses. Manyof the vesiculoviruses that infect mammals have been isolated fromnaturally infected arthropods, suggesting that they are vector-borne.Antibodies to vesiculoviruses are common among people living in ruralareas where the viruses are endemic and laboratory-acquired; infectionsin humans usually result in influenza-like symptoms. The Cocal virusenvelope glycoprotein shares 71.5% identity at the amino acid level withVSV-G Indiana, and phylogenetic comparison of the envelope gene ofvesiculoviruses shows that Cocal virus is serologically distinct from,but most closely related to, VSV-G Indiana strains among thevesiculoviruses. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) andTravassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999-1006(1984). The Cocal vesiculovirus envelope pseudotyped retroviral vectorparticles may include for example, lentiviral, alpharetroviral,betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviralvector particles that may comprise retroviral Gag, Pol, and/or one ormore accessory protein(s) and a Cocal vesiculovirus envelope protein.Within certain aspects of these embodiments, the Gag, Pol, and accessoryproteins are lentiviral and/or gammaretroviral. The invention providesAAV that contains or consists essentially of an exogenous nucleic acidmolecule encoding a CRISPR system, e.g., a plurality of cassettescomprising or consisting a first cassette comprising or consistingessentially of a promoter, a nucleic acid molecule encoding aCRISPR-associated (Cas) protein (putative nuclease or helicaseproteins), e.g., Cpf1 and a terminator, and a two, or more,advantageously up to the packaging size limit of the vector, e.g., intotal (including the first cassette) five, cassettes comprising orconsisting essentially of a promoter, nucleic acid molecule encodingguide RNA (gRNA) and a terminator (e.g., each cassette schematicallyrepresented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminatorPromoter-gRNA(N)-terminator (where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector), ortwo or more individual rAAVs, each containing one or more than onecassette of a CRISPR system, e.g., a first rAAV containing the firstcassette comprising or consisting essentially of a promoter, a nucleicacid molecule encoding Cas, e.g., Cas (Cpf1) and a terminator, and asecond rAAV containing a plurality, four, cassettes comprising orconsisting essentially of a promoter, nucleic acid molecule encodingguide RNA (gRNA) and a terminator (e.g., each cassette schematicallyrepresented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminatorPromoter-gRNA(N)-terminator (where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector). AsrAAV is a DNA virus, the nucleic acid molecules in the herein discussionconcerning AAV or rAAV are advantageously DNA. The promoter is in someembodiments advantageously human Synapsin I promoter (hSyn). Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr −/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepalc1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a CRISPR system as described herein (such as by transienttransfection of one or more vectors, or transfection with RNA), andmodified through the activity of a CRISPR complex, is used to establisha new cell line comprising cells containing the modification but lackingany other exogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors described herein,or cell lines derived from such cells are used in assessing one or moretest compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. Methods for producing transgenic animals and plants are known inthe art, and generally begin with a method of cell transfection, such asdescribed herein. In another embodiment, a fluid delivery device with anarray of needles (see, e.g., US Patent Publication No. 20110230839assigned to the Fred Hutchinson Cancer Research Center) may becontemplated for delivery of CRISPR Cas to solid tissue. A device of USPatent Publication No. 20110230839 for delivery of a fluid to a solidtissue may comprise a plurality of needles arranged in an array; aplurality of reservoirs, each in fluid communication with a respectiveone of the plurality of needles; and a plurality of actuatorsoperatively coupled to respective ones of the plurality of reservoirsand configured to control a fluid pressure within the reservoir. Incertain embodiments each of the plurality of actuators may comprise oneof a plurality of plungers, a first end of each of the plurality ofplungers being received in a respective one of the plurality ofreservoirs, and in certain further embodiments the plungers of theplurality of plungers are operatively coupled together at respectivesecond ends so as to be simultaneously depressable. Certain stillfurther embodiments may comprise a plunger driver configured to depressall of the plurality of plungers at a selectively variable rate. Inother embodiments each of the plurality of actuators may comprise one ofa plurality of fluid transmission lines having first and second ends, afirst end of each of the plurality of fluid transmission lines beingcoupled to a respective one of the plurality of reservoirs. In otherembodiments the device may comprise a fluid pressure source, and each ofthe plurality of actuators comprises a fluid coupling between the fluidpressure source and a respective one of the plurality of reservoirs. Infurther embodiments the fluid pressure source may comprise at least oneof a compressor, a vacuum accumulator, a peristaltic pump, a mastercylinder, a microfluidic pump, and a valve. In another embodiment, eachof the plurality of needles may comprise a plurality of portsdistributed along its length.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a nucleic acid-targeting complex to bind to thetarget polynucleotide to effect cleavage of said target polynucleotidethereby modifying the target polynucleotide, wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a nucleic acid-targeting complex to bind tothe polynucleotide such that said binding results in increased ordecreased expression of said polynucleotide; wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said polynucleotide.

CRISPR complex components may be delivered by conjugation or associationwith transport moieties (adapted for example from approaches disclosedin U.S. Pat. Nos. 8,106,022; 8,313,772). Nucleic acid deliverystrategies may for example be used to improve delivery of guide RNA, ormessenger RNAs or coding DNAs encoding CRISPR complex components. Forexample, RNAs may incorporate modified RNA nucleotides to improvestability, reduce immunostimulation, and/or improve specificity (seeDeleavey, Glen F. et al., 2012, Chemistry & Biology, Volume 19, Issue 8,937-954; Zalipsky, 1995, Advanced Drug Delivery Reviews 16: 157-182;Caliceti and Veronese, 2003, Advanced Drug Delivery Reviews 55:1261-1277). Various constructs have been described that may be used tomodify nucleic acids, such as gRNAs, for more efficient delivery, suchas reversible charge-neutralizing phosphotriester backbone modificationsthat may be adapted to modify gRNAs so as to be more hydrophobic andnon-anionic, thereby improving cell entry (Meade B R et al., 2014,Nature Biotechnology 32, 1256-1261). In further alternative embodiments,selected RNA motifs may be useful for mediating cellular transfection(Magalhães M., et al., Molecular Therapy (2012); 20 3, 616-624).Similarly, aptamers may be adapted for delivery of CRISPR complexcomponents, for example by appending aptamers to gRNAs (Tan W. et al.,2011, Trends in Biotechnology, December 2011, Vol. 29, No. 12).

In some embodiments, conjugation of triantennary N-acetyl galactosamine(GalNAc) to oligonucleotide components may be used to improve delivery,for example delivery to select cell types, for example hepatocytes (seeWO2014118272 incorporated herein by reference; Nair, J K et al., 2014,Journal of the American Chemical Society 136 (49), 16958-16961). Thismay be is considered to be a sugar-based particle and further details onother particle delivery systems and/or formulations are provided herein.GalNAc can therefore be considered to be a particle in the sense of theother particles described herein, such that general uses and otherconsiderations, for instance delivery of said particles, apply to GalNAcparticles as well. A solution-phase conjugation strategy may for examplebe used to attach triantennary GalNAc clusters (mol. wt. ˜2000)activated as PFP (pentafluorophenyl) esters onto 5′-hexylamino modifiedoligonucleotides (5′-HA ASOs, mol. wt. ˜8000 Da; Østergaard et al.,Bioconjugate Chem., 2015, 26 (8), pp 1451-1455). Similarly,poly(acrylate) polymers have been described for in vivo nucleic aciddelivery (see WO2013158141 incorporated herein by reference). In furtheralternative embodiments, pre-mixing CRISPR nanoparticles (or proteincomplexes) with naturally occurring serum proteins may be used in orderto improve delivery (Akinc A et al, 2010, Molecular Therapy vol. 18 no.7, 1357-1364).

Screening techniques are available to identify delivery enhancers, forexample by screening chemical libraries (Gilleron J. et al., 2015, Nucl.Acids Res. 43 (16): 7984-8001). Approaches have also been described forassessing the efficiency of delivery vehicles, such as lipidnanoparticles, which may be employed to identify effective deliveryvehicles for CRISPR components (see Sahay G. et al., 2013, NatureBiotechnology 31, 653-658).

In some embodiments, delivery of protein CRISPR components may befacilitated with the addition of functional peptides to the protein,such as peptides that change protein hydrophobicity, for example so asto improve in vivo functionality. CRISPR component proteins maysimilarly be modified to facilitate subsequent chemical reactions. Forexample, amino acids may be added to a protein that have a group thatundergoes click chemistry (Nikić I. et al., 2015, Nature Protocols10,780-791). In embodiments of this kind, the click chemical group maythen be used to add a wide variety of alternative structures, such aspoly(ethylene glycol) for stability, cell penetrating peptides, RNAaptamers, lipids, or carbohydrates such as GalNAc. In furtheralternatives, a CRISPR component protein may be modified to adapt theprotein for cell entry (see Svensen et al., 2012, Trends inPharmacological Sciences, Vol. 33, No. 4), for example by adding cellpenetrating peptides to the protein (see Kauffman, W. Berkeley et al.,2015, Trends in Biochemical Sciences, Volume 40, Issue 12, 749-764;Koren and Torchilin, 2012, Trends in Molecular Medicine, Vol. 18, No.7). In further alternative embodiment, patients or subjects may bepre-treated with compounds or formulations that facilitate the laterdelivery of CRISPR components.

Cpf1 Effector Protein Complexes can be Used in Plants

The Cpf1 effector protein system(s) (e.g., single or multiplexed) can beused in conjunction with recent advances in crop genomics. The systemsdescribed herein can be used to perform efficient and cost effectiveplant gene or genome interrogation or editing or manipulation—forinstance, for rapid investigation and/or selection and/or interrogationsand/or comparison and/or manipulations and/or transformation of plantgenes or genomes; e.g., to create, identify, develop, optimize, orconfer trait(s) or characteristic(s) to plant(s) or to transform a plantgenome. There can accordingly be improved production of plants, newplants with new combinations of traits or characteristics or new plantswith enhanced traits. The Cpf1 effector protein system(s) can be usedwith regard to plants in Site-Directed Integration (SDI) or Gene Editing(GE) or any Near Reverse Breeding (NRB) or Reverse Breeding (RB)techniques. Aspects of utilizing the herein described Cpf1 effectorprotein systems may be analogous to the use of the CRISPR-Cas (e.g.CRISPR-Cas9) system in plants, and mention is made of the University ofArizona website “CRISPR-PLANT” (http:www.genenome.arizona.edu/crisper/)(supported by Penn State and AGI). Embodiments of the invention can beused in genome editing in plants or where RNAi or similar genome editingtechniques have been used previously; see, e.g., Nekrasov, “Plant genomeediting made easy: targeted mutagenesis in model and crop plants usingthe CRISPR-Cas system,” Plant Methods 2013, 9:39(doi:10.1186/1746-4811-9-39); Brooks, “Efficient gene editing in tomatoin the first generation using the CRISPR-Cas9 system,” Plant PhysiologySeptember 2014 pp 114.247577; Shan, “Targeted genome modification ofcrop plants using a CRISPR-Cas system,” Nature Biotechnology 31, 686-688(2013); Feng, “Efficient genome editing in plants using a CRISPR/Cassystem,” Cell Research (2013) 23:1229-1232. doi:10.1038/cr.2013.114;published online 20 Aug. 2013; Xie, “RNA-guided genome editing in plantsusing a CRISPR-Cas system,” Mol Plant. 2013 November; 6(6):1975-83. doi:10.1093/mp/sst119. Epub 2013 Aug. 17; Xu, “Gene targeting using theAgrobacterium tumefaciens-mediated CRISPR-Cas system in rice,” Rice2014, 7:5 (2014), Zhou et al., “Exploiting SNPs for biallelic CRISPRmutations in the outcrossing woody perennial Populus reveals4-coumarate: CoA ligase specificity and Redundancy,” New Phytologist(2015) (Forum) 1-4 (available online only at www.newphytologist.com);Caliando et al, “Targeted DNA degradation using a CRISPR device stablycarried in the host genome, NATURE COMMUNICATIONS 6:6989, DOI:10.1038/ncomms7989, www.nature.com/naturecommunications DOI:10.1038/ncomms7989; U.S. Pat. No. 6,603,061—Agrobacterium-Mediated PlantTransformation Method; U.S. Pat. No. 7,868,149—Plant Genome Sequencesand Uses Thereof and US 2009/0100536—Transgenic Plants with EnhancedAgronomic Traits, all the contents and disclosure of each of which areherein incorporated by reference in their entirety. In the practice ofthe invention, the contents and disclosure of Morrell et al “Cropgenomics: advances and applications,” Nat Rev Genet. 2011 Dec. 29;13(2):85-96; each of which is incorporated by reference herein includingas to how herein embodiments may be used as to plants. Accordingly,reference herein to animal cells may also apply, mutatis mutandis, toplant cells unless otherwise apparent; and, the enzymes herein havingreduced off-target effects and systems employing such enzymes can beused in plant applications, including those mentioned herein.

Application of Cpf1-CRISPR System to Plants and Yeast Definitions

in general, the term “plant” relates to any various photosynthetic,eukaryotic, unicellular or multicellular organism of the kingdom Plantaecharacteristically growing by cell division, containing chloroplasts,and having cell walls comprised of cellulose. The term plant encompassesmonocotyledonous and dicotyledonous plants. Specifically, the plants areintended to comprise without limitation angiosperm and gymnosperm plantssuch as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree,asparagus, avocado, banana, barley, beans, beet, birch, beech,blackberry, blueberry, broccoli, Brussel's sprouts, cabbage, canola,cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery,chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee,corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive,eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts,ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm,okra, onion, orange, an ornamental plant or flower or tree, papaya,palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper,persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate,potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye,sorghum, safflower, sallow, soybean, spinach, spruce, squash,strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn,tangerine, tea, tobacco, tomato, trees, triticale, turf grasses,turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, andzucchini. The term plant also encompasses Algae, which are mainlyphotoautotrophs unified primarily by their lack of roots, leaves andother organs that characterize higher plants.

The methods for genome editing using the Cpf1 system as described hereincan be used to confer desired traits on essentially any plant. A widevariety of plants and plant cell systems may be engineered for thedesired physiological and agronomic characteristics described hereinusing the nucleic acid constructs of the present disclosure and thevarious transformation methods mentioned above. In preferredembodiments, target plants and plant cells for engineering include, hutare not limited to, those monocotyledonous and dicotyledonous plants,such as crops including grain crops (e.g., wheat, maize, rice, millet,barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange),forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot,potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce,spinach); flowering plants (e.g., petunia, rose, chrysanthemum),conifers and pine trees (e.g., pine fir, spruce); plants used inphytoremediation (e.g., heavy metal accumulating plants); oil crops(e.g., sunflower, rape seed) and plants used for experimental purposes(e.g., Arabidopsis). Thus, the methods and CRISPR-Cas systems can beused over a broad range of plants, such as for example withdicotyledonous plants belonging to the orders Magniolales, Illiciales,Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales,Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales,Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales,Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales,Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales,Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales,Myrtales, Cornales, Proteales, San tales, Rafflesiales, Celastrales,Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales,Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales,Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, andAsterales; the methods and CRISPR-Cas systems can be used withmonocotyledonous plants such as those belonging to the ordersAlismatales, Hydrocharitales, Najadales, Triuridales, Commelinales,Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,Lilliales, and Orchid ales, or with plants belonging to Gymnospermae,e.g those belonging to the orders Pinales, Ginkgoales, Cycadales,Araucariales, Cupressales and Gnetales.

The Cpf1 CRISPR systems and methods of use described herein can be usedover a broad range of plant species, included in the non-limitative listof dicot, monocot or gymnosperm genera hereunder: Atropa, Alseodaphne,Anacardium, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus,Croton, Cucumis, Citrus, Citrullus, Capsicum, Catharanthus, Cocos,Coffea, Cucurbita, Daucus, Duguelia, Eschscholzia, Ficus, Fragaria,Glaucium, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca,Landolphia, Liinum, Litsea, Lycopersicon, Lupinus, Manihot, Majorana,Malus, Medicago, Nicoliana, Olea, Parthenium, Papaver, Persea,Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphans, Ricirms, Senecio,Sinomenium, Stephania, Sinapis, Solanum, Theobroma, Trifolium,Trigonella, Vicia, Vinca, Vilis and Vigna; and the genera Allium,Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca,Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum,Pannesetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies,Cunninghamia, Ephedra, Picea, Pinus, and Pseudoisuga.

The Cpf1 CRISPR systems and methods of use can also be used over a broadrange of “algae” or “algae cells”; including for example algae selectedfrom several eukaryotic phyla, including the Rhodophyta (red algae),Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta(diatoms), Eustigmatophyta and dinoflagellates as well as theprokaryotic phylum Cyanobacteria (blue-green algae). The term “algae”includes for example algae selected from: Amphora, Anabaena,Anikstrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella,Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena,Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris,Nannnochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia,Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Pavlova,Phaeodactylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena,Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis,Thalassiosira, and Trichodesmium.

A part of a plant, i.e., a “plant tissue” may be treated according tothe methods of the present invention to produce an improved plant. Planttissue also encompasses plant cells. The term “plant cell” as usedherein refers to individual units of a living plant, either in an intactwhole plant or in an isolated form grown in in vitro tissue cultures, onmedia or agar, in suspension in a growth media or buffer or as a part ofhigher organized unites, such as, for example, plant tissue, a plantorgan, or a whole plant.

A “protoplast” refers to a plant cell that has had its protective cellwall completely or partially removed using, for example, mechanical orenzymatic means resulting in an intact biochemical competent unit ofliving plant that can reform their cell wall, proliferate and regenerategrow into a whole plant under proper growing conditions.

The term “transformation” broadly refers to the process by which a planthost is genetically modified by the introduction of DNA by means ofAgrobacteria or one of a variety of chemical or physical methods. Asused herein, the term “plant host” refers to plants, including anycells, tissues, organs, or progeny of the plants. Many suitable planttissues or plant cells can be transformed and include, but are notlimited to, protoplasts, somatic embryos, pollen, leaves, seedlings,stems, calli, stolons, microtubers, and shoots. A plant tissue alsorefers to any clone of such a plant, seed, progeny, propagule whethergenerated sexually or asexually, and descendents of any of these, suchas cuttings or seed.

The term “transformed” as used herein, refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is transmitted to the subsequent progeny. Inthese embodiments, the “transformed” or “transgenic” cell or plant mayalso include progeny of the cell or plant and progeny produced from abreeding program employing such a transformed plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe introduced DNA molecule. Preferably, the transgenic plant is fertileand capable of transmitting the introduced DNA to progeny through sexualreproduction.

The term “progeny”, such as the progeny of a transgenic plant, is onethat is born of, begotten by, or derived from a plant or the transgenicplant. The introduced DNA molecule may also be transiently introducedinto the recipient cell such that the introduced DNA molecule is notinherited by subsequent progeny and thus not considered “transgenic”.Accordingly, as used herein, a “non-transgenic” plant or plant cell is aplant which does not contain a foreign DNA stably integrated into itsgenome.

The term “plant promoter” as used herein is a promoter capable ofinitiating transcription in plant cells, whether or not its origin is aplant cell. Exemplary suitable plant promoters include, but are notlimited to, those that are obtained from plants, plant viruses, andbacteria such as Agrobacterium or Rhizobium which comprise genesexpressed in plant cells.

As used herein, a “fungal cell” refers to any type of eukaryotic cellwithin the kingdom of fungi. Phyla within the kingdom of fungi includeAscomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota,Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cellsmay include yeasts, molds, and filamentous fungi. In some embodiments,the fungal cell is a yeast cell.

As used herein, the term “yeast cell” refers to any fungal cell withinthe phyla Ascomycota and Basidiomycota. Yeast cells may include buddingyeast cells, fission yeast cells, and mold cells. Without being limitedto these organisms, many types of yeast used in laboratory andindustrial settings are part of the phylum Ascomycota. In someembodiments, the yeast cell is an S. cerervisiae, Kluyveromycesmarxianus, or Issatchenkia orientalis cell. Other yeast cells mayinclude without limitation Candida spp. (e.g., Candida albicans),Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichiapastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis andKluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa),Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g.,Issatchenkia orientalis, a.k.a. Pichia kudriavzevii and Candidaacidothermophilum). In some embodiments, the fungal cell is afilamentous fungal cell. As used herein, the term “filamentous fungalcell” refers to any type of fungal cell that grows in filaments, i.e.,hyphae or mycelia. Examples of filamentous fungal cells may includewithout limitation Aspergillus spp. (e.g., Aspergillus niger),Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g.,Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

In some embodiments, the fungal cell is an industrial strain. As usedherein, “industrial strain” refers to any strain of fungal cell used inor isolated from an industrial process, e.g., production of a product ona commercial or industrial scale. Industrial strain may refer to afungal species that is typically used in an industrial process, or itmay refer to an isolate of a fungal species that may be also used fornon-industrial purposes (e.g., laboratory research). Examples ofindustrial processes may include fermentation (e.g., in production offood or beverage products), distillation, biofuel production, productionof a compound, and production of a polypeptide. Examples of industrialstrains may include, without limitation, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As usedherein, a “polyploid” cell may refer to any cell whose genome is presentin more than one copy. A polyploid cell may refer to a type of cell thatis naturally found in a polyploid state, or it may refer to a cell thathas been induced to exist in a polyploid state (e.g., through specificregulation, alteration, inactivation, activation, or modification ofmeiosis, cytokinesis, or DNA replication). A polyploid cell may refer toa cell whose entire genome is polyploid, or it may refer to a cell thatis polyploid in a particular genomic locus of interest. Without wishingto be bound to theory, it is thought that the abundance of guideRNA maymore often be a rate-limiting component in genome engineering ofpolyploid cells than in haploid cells, and thus the methods using theCpf1 CRISPRS system described herein may take advantage of using acertain fungal cell type.

In some embodiments, the fungal cell is a diploid cell. As used herein,a “diploid” cell may refer to any cell whose genome is present in twocopies. A diploid cell may refer to a type of cell that is naturallyfound in a diploid state, or it may refer to a cell that has beeninduced to exist in a diploid state (e.g., through specific regulation,alteration, inactivation, activation, or modification of meiosis,cytokinesis, or DNA replication). For example, the S. cerevisiae strainS228C may be maintained in a haploid or diploid state. A diploid cellmay refer to a cell whose entire genome is diploid, or it may refer to acell that is diploid in a particular genomic locus of interest. In someembodiments, the fungal cell is a haploid cell. As used herein, a“haploid” cell may refer to any cell whose genome is present in onecopy. A haploid cell may refer to a type of cell that is naturally foundin a haploid state, or it may refer to a cell that has been induced toexist in a haploid state (e.g., through specific regulation, alteration,inactivation, activation, or modification of meiosis, cytokinesis, orDNA replication). For example, the S. cerevisiae strain S228C may bemaintained in a haploid or diploid state. A haploid cell may refer to acell whose entire genome is haploid, or it may refer to a cell that ishaploid in a particular genomic locus of interest.

As used herein, a “yeast expression vector” refers to a nucleic acidthat contains one or more sequences encoding an RNA and/or polypeptideand may further contain any desired elements that control the expressionof the nucleic acid(s), as well as any elements that enable thereplication and maintenance of the expression vector inside the yeastcell. Many suitable yeast expression vectors and features thereof areknown in the art; for example, various vectors and techniques areillustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (HumanaPress, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991)Biotechnology (NY) 9(11): 1067-72. Yeast vectors may contain, withoutlimitation, a centromeric (CEN) sequence, an autonomous replicationsequence (ARS), a promoter, such as an RNA Polymerase III promoter,operably linked to a sequence or gene of interest, a terminator such asan RNA polymerase III terminator, an origin of replication, and a markergene (e.g., auxotrophic, antibiotic, or other selectable markers).Examples of expression vectors for use in yeast may include plasmids,yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids,yeast replicative plasmids, shuttle vectors, and episomal plasmids.

Stable Integration of Cpf1 CRISP System Components in the Genome ofPlants and Plant Cells

In particular embodiments, it is envisaged that the polynucleotidesencoding the components of the Cpf1 CRISPR system are introduced forstable integration into the genome of a plant cell. In theseembodiments, the design of the transformation vector or the expressionsystem can be adjusted depending on for when, where and under whatconditions the guide RNA and/or the Cpf1 gene are expressed.

In particular embodiments, it is envisaged to introduce the componentsof the Cpf1 CRISPR system stably into the genomic DNA of a plant cell.Additionally or alternatively, it is envisaged to introduce thecomponents of the Cpf1 CRISPR system for stable integration into the DNAof a plant organelle such as, but not limited to a plastid, emitochondrion or a chloroplast.

The expression system for stable integration into the genome of a plantcell may contain one or more of the following elements: a promoterelement that can be used to express the RNA and/or Cpf1 enzyme in aplant cell; a 5′ untranslated region to enhance expression; an intronelement to further enhance expression in certain cells, such as monocotcells; a multiple-cloning site to provide convenient restriction sitesfor inserting the guide RNA and/or the Cpf1 gene sequences and otherdesired elements; and a 3′ untranslated region to provide for efficienttermination of the expressed transcript.

The elements of the expression system may be on one or more expressionconstructs which are either circular such as a plasmid or transformationvector, or non-circular such as linear double stranded DNA.

In a particular embodiment, a Cfp1 CRISPR expression system comprises atleast:

-   (a) a nucleotide sequence encoding a guide RNA (gRNA) that    hybridizes with a target sequence in a plant, and wherein the guide    RNA comprises a guide sequence and a direct repeat sequence, and-   (b) a nucleotide sequence encoding a Cpf1 protein,    wherein components (a) or (b) are located on the same or on    different constructs, and whereby the different nucleotide sequences    can be under control of the same or a different regulatory element    operable in a plant cell.

DNA construct(s) containing the components of the Cpf1 CRISPR system,and, where applicable, template sequence may be introduced into thegenome of a plant, plant part, or plant cell by a variety ofconventional techniques. The process generally comprises the steps ofselecting a suitable host cell or host tissue, introducing theconstruct(s) into the host cell or host tissue, and regenerating plantcells or plants therefrom.

In particular embodiments, the DNA construct nay be introduced into theplant cell using techniques such as but not limited to electroporation,microinjection, aerosol beam injection of plant cell protoplasts, or theDNA constructs can be introduced directly to plant tissue usingbiolistic methods, such as DNA particle bombardment (see also Fu et al.,Transgenic Res. 2000 February; 9(1):11-9). The basis of particlebombardment is the acceleration of particles coated with gene/s ofinterest toward cells, resulting in the penetration of the protoplasm bythe particles and typically stable integration into the genome. (seee.g. Klein et al, Nature (1987), Klein et ah, Bio/Technology (1992),Casas et ah, Proc. Natl. Acad. Sci. USA (1993)).

In particular embodiments, the DNA constructs containing components ofthe Cpf1 CRISPR system may be introduced into the plant byAgrobacterium-mediated transformation. The DNA constructs may becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The foreign DNA canbe incorporated into the genome of plants by infecting the plants or byincubating plant protoplasts with Agrobacterium bacteria, containing oneor more Ti (tumor-inducing) plasmids. (see e.g. Fraley et al., (1985),Rogers et al., (1987) and U.S. Pat. No. 5,563,055).

Plant Promoters

In order to ensure appropriate expression in a plant cell, thecomponents of the Cpf1 CRISPR system described herein are typicallyplaced under control of a plant promoter, i.e. a promoter operable inplant cells. The use of different types of promoters is envisaged.

A constitutive plant promoter is a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of the plant(referred to as “constitutive expression”). One non-limiting example ofa constitutive promoter is the cauliflower mosaic virus 358 promoter.“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and includes tissue-specific, tissue-preferred and inducible promoters.Different promoters may direct the expression of a gene in differenttissues or cell types, or at different stages of development, or inresponse to different environmental conditions. In particularembodiments, one or more of the Cpf1 CRISPR components are expressedunder the control of a constitutive promoter, such as the cauliflowermosaic virus 35S promoter issue-preferred promoters can be utilized totarget enhanced expression in certain cell types within a particularplant tissue, for instance vascular cells in leaves or roots or inspecific cells of the seed. Examples of particular promoters for use inthe Cpf1 CRISPR system- are found in Kawamata et al., (1997) Plant CellPhysiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire etal, (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681-91.

Examples of promoters that are inducible and that allow forspatiotemporal control of gene editing or gene expression may use a formof energy. The form of energy may include but is not limited to soundenergy, electromagnetic radiation, chemical energy and/or thermalenergy. Examples of inducible systems include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome)., such as a Light InducibleTranscriptional Effector (LITE) that direct changes in transcriptionalactivity in a sequence-specific manner. The components of a lightinducible system may include a Cpf1 CRISPR enzyme, a light-responsivecytochrome heterodimer (e.g. from Arabidopsis thaliana), and atranscriptional activation/repression domain. Further examples ofinducible DNA binding proteins and methods for their use are provided inU.S. 61/736,465 and U.S. 61/721,283, which is hereby incorporated byreference in its entirety.

In particular embodiments, transient or inducible expression can beachieved by using, for example, chemical-regulated promotors, i.e.whereby the application of an exogenous chemical induces geneexpression. Modulating of gene expression can also be obtained by achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters include, but arenot limited to, the maize ln2-2 promoter, activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant CellPhysiol 38:568-77), the maize GST promoter (GST-ll-27, WO93/01294),activated by hydrophobic electrophilic compounds used as pre-emergentherbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) BiosciBiotechnol Biochem 68:803-7) activated by salicylic acid. Promoterswhich are regulated by antibiotics, such as tetracycline-inducible andtetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be usedherein.

Translocation to and/or Expression in Specific Plant Organelles

The expression system may comprise elements for translocation to and/orexpression in a specific plant organelle.

Chloroplast Targeting

In particular embodiments, it is envisaged that the Cpf1 CRISPR systemis used to specifically modify chloroplast genes or to ensure expressionin the chloroplast. For this purpose use is made of chloroplasttransformation methods or compartimentalization of the Cpf1 CRISPRcomponents to the chloroplast. For instance, the introduction of geneticmodifications in the plastid genome can reduce biosafety issues such asgene flow through pollen.

Methods of chloroplast transformation are known in the art and includeParticle bombardment, PEG treatment, and microinjection. Additionally,methods involving the translocation of transformation cassettes from thenuclear genome to the pastid can be used as described in WO2010061186.

Alternatively, it is envisaged to target one or more of the Cpf1 CRISPRcomponents to the plant chloroplast. This is achieved by incorporatingin the expression construct a sequence encoding a chloroplast transitpeptide (CTP) or plastid transit peptide, operably linked to the 5′region of the sequence encoding the Cpf1 protein. The CTP is removed ina processing step during translocation into the chloroplast. Chloroplasttargeting of expressed proteins is well known to the skilled artisan(see for instance Protein Transport into Chloroplasts, 2010, AnnualReview of Plant Biology, Vol, 61: 157-180). In such embodiments it isalso desired to target the guide RNA to the plant chloroplast. Methodsand constructs which can be used for translocating guide RNA into thechloroplast by means of a chloroplast localization sequence aredescribed, for instance, in US 20040142476, incorporated herein byreference. Such variations of constructs can be incorporated into theexpression systems of the invention to efficiently translocate theCpf1-guide RNA.

Introduction of Polynucleotides Encoding the CRISPR-Cpf1 System in AlgalCells.

Transgenic algae (or other plants such as rape) may be particularlyuseful in the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol) or other products. These may beengineered to express or overexpress high levels of oil or alcohols foruse in the oil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the Cpf1 CRISPR system described herein can beapplied on Chlamydomonas species and other algae. In particularembodiments, Cpf1 and guide RNA are introduced in algae expressed usinga vector that expresses Cpf1 under the control of a constitutivepromoter such as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA is optionallydelivered using a vector containing T7 promoter. Alternatively, Cas9mRNA and in vitro transcribed guide RNA can be delivered to algal cells.Electroporation protocols are available to the skilled person such asthe standard recommended protocol from the GeneArt ChlamydomonasEngineering kit.

In particular embodiments, the endonuclease used herein is a Split Cpf1enzyme. Split Cpf1 enzymes are preferentially used in Algae for targetedgenome modification as has been described for Cas9 in WO 2015086795. Useof the Cpf1 split system is particularly suitable for an induciblemethod of genome targeting and avoids the potential toxic effect of theCpf1 overexpression within the algae cell. In particular embodiments,Said Cpf1 split domains (RuvC and HNH domains) can be simultaneously orsequentially introduced into the cell such that said split Cpf1domain(s) process the target nucleic acid sequence in the algae cell.The reduced size of the split Cpf1 compared to the wild type Cpf1 allowsother methods of delivery of the CRISPR system to the cells, such as theuse of Cell Penetrating Peptides as described herein. This method is ofparticular interest for generating genetically modified algae.

Introduction of Polynucleotides Encoding Cpf1 Components in Yeast Cells

In particular embodiments, the invention relates to the use of the Cpf1CRISPR system for genome editing of yeast cells. Methods fortransforming yeast cells which can be used to introduce polynucleotidesencoding the Cpf1 CRISPR system components are well known to the artisanand are reviewed by Kawai et al., 2010, Bioeng Bugs. 2010November-December; 1(6): 395-403). Non-limiting examples includetransformation of yeast cells by lithium acetate treatment (which mayfurther include carrier DNA and PEG treatment), bombardment or byelectroporation.

Transient Expression of Cpf1 CRISP System Components in Plants and PlantCell

In particular embodiments, it is envisaged that the guide RNA and/orCpf1 gene are transiently expressed in the plant cell. In theseembodiments, the Cpf1 CRISPR system can ensure modification of a targetgene only when both the guide RNA and the Cpf1 protein is present in acell, such that genomic modification can further be controlled. As theexpression of the Cpf1 enzyme is transient, plants regenerated from suchplant cells typically contain no foreign DNA. In particular embodimentsthe Cpf1 enzyme is stably expressed by the plant cell and the guidesequence is transiently expressed.

In particular embodiments, the Cpf1 CRISPR system components can beintroduced in the plant cells using a plant viral vector (Scholthof etal. 1996, Annu. Rev Phytopathol. 1996; 34:299-323). In furtherparticular embodiments, said viral vector is a vector from a DNA virus.For example, geminivirus (e.g., cabbage leaf curl virus, bean yellowdwarf virus, wheat dwarf virus, tomato leaf curl virus, maize streakvirus, tobacco leaf curl virus, or tomato golden mosaic virus) ornanovirus (e.g., Faba bean necrotic yellow virus). In other particularembodiments, said viral vector is a vector from an RNA virus. Forexample, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus),potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripemosaic virus). The replicating genomes of plant viruses arenon-integrative vectors.

In particular embodiments, the vector used for transient expression ofCpf1 CRISPR constructs is for instance a pEAQ vector, which is tailoredfor Agrobacterium-mediated transient expression (Sainsbury F, et al.,Plant. Biotechnol J. 2009 September; 7(7):682-93) in the protoplast.Precise targeting of genomic locations was demonstrated using amodified. Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs instable transgenic plants expressing a CRISPR enzyme (Scientific Reports5, Article number: 14926 (2015), doi:10.1038/srep14926).

In particular embodiments, double-stranded DNA fragments encoding theguide RNA and/or the Cpf1 gene can be transiently introduced into theplant cell. In such embodiments, the introduced double-stranded DNAfragments are provided in sufficient quantity to modify the cell but donot persist after a contemplated period of time has passed or after oneor more cell divisions. Methods for direct DNA transfer in plants areknown by the skilled artisan (see for instance Davey et al, Plant MolBiol. 1989 September; 13(3):273-85.)

In other embodiments, an RNA polynucleotide encoding the Cpf1 protein isintroduced into the plant cell, which is then translated and processedby the host cell generating the protein in sufficient quantity to modifythe cell (in the presence of at least one guide RNA) but which does notpersist after a contemplated period of time has passed or after one ormore cell divisions. Methods for introducing mRNA to plant protoplastsfor transient expression are known by the skilled artisan (see forinstance in Gallie, Plant Cell Reports (1993), 13; 119-122).

Combinations of the different methods described above are alsoenvisaged.

Delivery of Cpf1 CRISPR Components to the Plant Cell

In particular embodiments, it is of interest to deliver one or morecomponents of the Cpf1 CRISPR system directly to the plant cell. This isof interest, inter alia, for the generation of non-transgenic plants(see below). In particular embodiments, one or more of the Cpf1components is prepared outside the plant or plant cell and delivered tothe cell. For instance in particular embodiments, the Cpf1 protein isprepared in vitro prior to introduction to the plant cell. Cpf1 proteincan be prepared by various methods known by one of skill in the art andinclude recombinant production. After expression, the Cpf1 protein isisolated, refolded if needed, purified and optionally treated to removeany purification tags, such as a His-tag. Once crude, partiallypurified, or more completely purified Cpf1 protein is obtained, theprotein may be introduced to the plant cell.

In particular embodiments, the Cpf1 protein is mixed with guide RNAtargeting the gene of interest to form a pre-assembledribonucleoprotein.

The individual components or pre-assembled ribonucleoprotein can beintroduced into the plant cell via electroporation, by bombardment withCpf1-associated gene product coated particles, by chemical transfectionor by some other means of transport across a cell membrane. Forinstance, transfection of a plant protoplast with a pre-assembled CRISPRribonucleoprotein has been demonstrated to ensure targeted modificationof the plant genome (as described by Woo et al, Nature Biotechnology,2015; DOI: 10.1038/nbt.3389).

In particular embodiments, the Cpf1 CRISPR system components areintroduced into the plant cells using nanoparticles. The components,either as protein or nucleic acid or in a combination thereof, can beuploaded onto or packaged in nanoparticles and applied to the plants(such as for instance described in WO 2008042156 and US 20130185823). Inparticular, embodiments of the invention comprise nanoparticles uploadedwith or packed with DNA molecule(s) encoding the Cpf1 protein, DNAmolecules encoding the guide RNA and/or isolated guide RNA as describedin WO2015089419.

Further means of introducing one or more components of the Cpf1 CRISPRsystem to the plant cell is by using cell penetrating peptides (CPP).Accordingly, in particular, embodiments the invention comprisescompositions comprising a cell penetrating peptide linked to the Cpf1protein. In particular embodiments of the present invention, the Cpf1protein and/or guide RNA is coupled to one or more CPPs to effectivelytransport them inside plant protoplasts; see also Ramakrishna (20140Genome Res. 2014 June; 24(6):1020-7 for Cas9 in human cells). In otherembodiments, the Cpf1 gene and/or guide RNA are encoded by one or morecircular or non-circular DNA molecule(s) which are coupled to one ormore CPPs for plant protoplast delivery. The plant protoplasts are thenregenerated to plant cells and further to plants. CPPs are generallydescribed as short peptides of fewer than 35 amino acids either derivedfrom proteins or from chimeric sequences which are capable oftransporting biomolecules across cell membrane in a receptor independentmanner. CPP can be cationic peptides, peptides having hydrophobicsequences, amphipatic peptides, peptides having proline-rich andanti-microbial sequence, and chimeric or bipartite peptides (Pooga andLangel 2005). CPPs are able to penetrate biological membranes and assuch trigger the movement of various biomolecules across cell membranesinto the cytoplasm and to improve their intracellular routing, and hencefacilitate interaction of the biolomolecule with the target. Examples ofCPP include amongst others: Tat, a nuclear transcriptional activatorprotein required for viral replication by HIV type 1, penetratin, Kaposifibroblast growth factor (FGF) signal peptide sequence, integrin β3signal peptide sequence; polyarginine peptide Args sequence, Guaninerich-molecular transporters, sweet arrow peptide, etc. . . . .

Use of the Cpf1 CRISPR System to Make Genetically ModifiedNon-Transgenic Plants

In particular embodiments, the methods described herein are used tomodify endogenous genes or to modify their expression without thepermanent introduction into the genome of the plant of any foreign gene,including those encoding CRISPR components, so as to avoid the presenceof foreign DNA in the genome of the plant. This can be of interest asthe regulatory requirements for non-transgenic plants are less rigorous.

In particular embodiments, this is ensured by transient expression ofthe Cpf1 CRISPR components. In particular embodiments one or more of theCRISPR components are expressed on one or more viral vectors whichproduce sufficient Cpf1 protein and guide RNA to consistently steadilyensure modification of a gene of interest according to a methoddescribed herein.

In particular embodiments, transient expression of Cpf1 CRISPRconstructs is ensured in plant protoplasts and thus not integrated intothe genome. The limited window of expression can be sufficient to allowthe Cpf1 CRISPR system to ensure modification of a target gene asdescribed herein.

In particular embodiments, the different components of the Cpf1 CRISPRsystem are introduced in the plant cell, protoplast or plant tissueeither separately or in mixture, with the aid of particulate deliveringmolecules such as nanoparticles or CPP molecules as described hereinabove.

The expression of the Cpf1 CRISPR components can induce targetedmodification of the genome, either by direct activity of the Cpf1nuclease and optionally introduction of template DNA or by modificationof genes targeted using the Cpf1 CRISPR system as described herein. Thedifferent strategies described herein above allow Cpf1-mediated targetedgenome editing without requiring the introduction of the Cpf1 CRISPRcomponents into the plant genome. Components which are transientlyintroduced into the plant cell are typically removed upon crossing.

Detecting Modifications in the Plant Genome-Selectable Markers

In particular embodiments, where the method involves modification of anendogeneous target gene of the plant genome, any suitable method can beused to determine, after the plant, plant part or plant cell is infectedor transfected with the Cpf1 CRISPR system, whether gene targeting ortargeted mutagenesis has occurred at the target site. Where the methodinvolves introduction of a transgene, a transformed plant cell, callus,tissue or plant may be identified and isolated by selecting or screeningthe engineered plant material for the presence of the transgene or fortraits encoded by the transgene. Physical and biochemical methods may beused to identify plant or plant cell transformants containing insertedgene constructs or an endogenous DNA modification. These methods includebut are not limited to: 1) Southern analysis or PCR amplification fordetecting and determining the structure of the recombinant DNA insert ormodified endogenous genes; 2) Northern blot, S1 RNase protection,primer-extension or reverse transcriptase-PCR amplification fordetecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct or expression isaffected by the genetic modification; 4) protein gel electrophoresis,Western blot techniques, immunoprecipitation, or enzyme-linkedimmunoassays, where the gene construct or endogenous gene products areproteins. Additional techniques, such as in situ hybridization, enzymestaining, and immunostaining, also may be used to detect the presence orexpression of the recombinant construct or detect a modification ofendogenous gene in specific plant organs and tissues. The methods fordoing all these assays are well known to those skilled in the art.

Additionally (or alternatively), the expression system encoding the Cpf1CRISPR components is typically designed to comprise one or moreselectable or detectable markers that provide a means to isolate orefficiently select cells that contain and/or have been modified by theCpf1 CRISPR system at an early stage and on a large scale.

In the case of Agrobacterium-mediated transformation, the markercassette may be adjacent to or between flanking T-DNA borders andcontained within a binary vector. In another embodiment, the markercassette may be outside of the T-DNA. A selectable marker cassette mayalso be within or adjacent to the same T-DNA borders as the expressioncassette or may be somewhere else within a second T-DNA on the binaryvector (e.g., a 2 T-DNA system).

For particle bombardment or with protoplast transformation, theexpression system can comprise one or more isolated linear fragments ormay be part of a larger construct that might contain bacterialreplication elements, bacterial selectable markers or other detectableelements. The expression cassette(s) comprising the polynucleotidesencoding the guide and/or Cpf1 may be physically linked to a markercassette or may be mixed with a second nucleic acid molecule encoding amarker cassette. The marker cassette is comprised of necessary elementsto express a detectable or selectable marker that allows for efficientselection of transformed cells.

The selection procedure for the cells based on the selectable markerwill depend on the nature of the marker gene. In particular embodiments,use is made of a selectable marker, i.e. a marker which allows a directselection of the cells based on the expression of the marker. Aselectable marker can confer positive or negative selection and isconditional or non-conditional on the presence of external substrates(Miki et al. 2004, 107(3): 193-232). Most commonly, antibiotic orherbicide resistance genes are used as a marker, whereby selection is beperformed by growing the engineered plant material on media containingan inhibitory amount of the antibiotic or herbicide to which the markergene confers resistance. Examples of such genes are genes that conferresistance to antibiotics, such as hygromycin (hpt) and kanamycin(nptII), and genes that confer resistance to herbicides, such asphosphinothricin (bar) and chlorosulfuron (als),

Transformed plants and plant cells may also be identified by screeningfor the activities of a visible marker, typically an enzyme capable ofprocessing a colored substrate (e.g., the β-glucuronidase, luciferase, Bor C1 genes). Such selection and screening methodologies are well knownto those skilled in the art.

Plant Cultures and Regeneration

In particular embodiments, plant cells which have a modified genome andthat are produced or obtained by any of the methods described herein,can be cultured to regenerate a whole plant which possesses thetransformed or modified genotype and thus the desired phenotype.Conventional regeneration techniques are well known to those skilled inthe art. Particular examples of such regeneration techniques rely onmanipulation of certain phytohormones in a tissue culture growth medium,and typically relying on a biocide and/or herbicide marker which hasbeen introduced together with the desired nucleotide sequences. Infurther particular embodiments, plant regeneration is obtained fromcultured protoplasts, plant callus, explants, organs, pollens, embryosor parts thereof (see e.g. Evans et al. (1983), Handbook of Plant CellCulture, Klee et al (1987) Ann. Rev. of Plant Phys.).

In particular embodiments, transformed or improved plants as describedherein can be self-pollinated to provide seed for homozygous improvedplants of the invention (homozygous for the DNA modification) or crossedwith non-transgenic plants or different improved plants to provide seedfor heterozygous plants. Where a recombinant DNA was introduced into theplant cell, the resulting plant of such a crossing is a plant which isheterozygous for the recombinant DNA molecule. Both such homozygous andheterozygous plants obtained by crossing from the improved plants andcomprising the genetic modification (which can be a recombinant DNA) arereferred to herein as “progeny”. Progeny plants are plants descendedfrom the original transgenic plant and containing the genomemodification or recombinant DNA molecule introduced by the methodsprovided herein. Alternatively, genetically modified plants can beobtained by one of the methods described supra using the Cfp1 enzymewhereby no foreign DNA is incorporated into the genome. Progeny of suchplants, obtained by further breeding may also contain the geneticmodification. Breedings are performed by any breeding methods that arecommonly used for different crops (e.g., Allard, Principles of PlantBreeding, John Wiley & Sons, NY, U. of CA, Davis, Calif., 50-98 (1960).

Generation of Plants with Enhanced Agronomic Traits

The Cpf1 based CRISPR systems provided herein can be used to introducetargeted double-strand or single-strand breaks and/or to introduce geneactivator and or repressor systems and without being (imitative, can beused for gene targeting, gene replacement, targeted mutagenesis,targeted deletions or insertions, targeted inversions and/or targetedtranslocations. By co-expression of multiple targeting RNAs directed toachieve multiple modifications in a single cell, multiplexed genomemodification can be ensured. This technology can be used tohigh-precision engineering of plants with improved characteristics,including enhanced nutritional quality, increased resistance to diseasesand resistance to biotic and abiotic stress, and increased production ofcommercially valuable plant products or heterologous compounds.

In particular embodiments, the Cpf1 CRISPR system as described herein isused to introduce targeted double-strand breaks (DSB) in an endogenousDNA sequence. The DSB activates cellular DNA repair pathways, which canbe harnessed to achieve desired DNA sequence modifications near thebreak site. This is of interest where the inactivation of endogenousgenes can confer or contribute to a desired trait. In particularembodiments, homologous recombination with a template sequence ispromoted at the site of the DSB, in order to introduce a gene ofinterest.

In particular embodiments, the Cpf1 CRISPR system may be used as ageneric nucleic acid binding protein with fusion to or being operablylinked to a functional domain for activation and/or repression ofendogenous plant genes. Exemplary functional domains may include but arenot limited to translational initiator, translational activator,translational repressor, nucleases, in particular ribonucleases, aspliceosome, beads, a light inducible/controllable domain or achemically inducible/controllable domain. Typically in theseembodiments, the Cpf1 protein comprises at least one mutation, such thatit has no more than 5% of the activity of the Cpf1 protein not havingthe at least one mutation; the guide RNA comprises a guide sequencecapable of hybridizing to a target sequence.

The methods described herein generally result in the generation of“improved plants” in that they have one or more desirable traitscompared to the wildtype plant. In particular embodiments, the plants,plant cells or plant parts obtained are transgenic plants, comprising anexogenous DNA sequence incorporated into the genome of all or part ofthe cells of the plant. In particular embodiments, non-transgenicgenetically modified plants, plant parts or cells are obtained, in thatno exogenous DNA sequence is incorporated into the genome of any of theplant cells of the plant. In such embodiments, the improved plants arenon-transgenic. Where only the modification of an endogenous gene isensured and no foreign genes are introduced or maintained in the plantgenome, the resulting genetically modified crops contain no foreigngenes and can thus basically be considered non-transgenic. The differentapplications of the Cpf1 CRISPR system for plant genome editing aredescribed more in detail below:

a) Introduction of One or More Foreign Genes to Confer an AgriculturalTrait of Interest

The invention provides methods of genome editing or modifying sequencesassociated with or at a target locus of interest wherein the methodcomprises introducing a Cpf1 effector protein complex into a plant cell,whereby the Cpf1 effector protein complex effectively functions tointegrate a DNA insert, e.g. encoding a foreign gene of interest, intothe genome of the plant cell. In preferred embodiments the integrationof the DNA insert is facilitated by HR with an exogenously introducedDNA template or repair template. Typically, the exogenously introducedDNA template or repair template is delivered together with the Cpf1effector protein complex or one component or a polynucleotide vector forexpression of a component of the complex.

The Cpf1 CRISPR systems provided herein allow for targeted genedelivery. It has become increasingly clear that the efficiency ofexpressing a gene of interest is to a great extent determined by thelocation of integration into the genome. The present methods allow fortargeted integration of the foreign gene into a desired location in thegenome. The location can be selected based on information of previouslygenerated events or can be selected by methods disclosed elsewhereherein.

In particular embodiments, the methods provided herein include (a)introducing into the cell a Cpf1 CRISPR complex comprising a guide RNA,comprising a direct repeat and a guide sequence, wherein the guidesequence hybrdizes to a target sequence that is endogenous to the plantcell; (b) introducing into the plant cell a Cpf1 effector molecule whichcomplexes with the guide RNA when the guide sequence hybridizes to thetarget sequence and induces a double strand break at or near thesequence to which the guide sequence is targeted; and (c) introducinginto the cell a nucleotide sequence encoding an HDR repair templatewhich encodes the gene of interest and which is introduced into thelocation of the DS break as a result of HDR. In particular embodiments,the step of introducing can include delivering to the plant cell one ormore polynucleotides encoding Cpf1 effector protein, the guide RNA andthe repair template. In particular embodiments, the polynucleotides aredelivered into the cell by a DNA virus (e.g., a geminivirus) or an RNAvirus (e.g., a tobravirus). In particular embodiments, the introducingsteps include delivering to the plant cell a T-DNA containing one ormore polynucleotide sequences encoding the Cpf1 effector protein, theguide RNA and the repair template, where the delivering is viaAgrobacterium. The nucleic acid sequence encoding the Cpf1 effectorprotein can be operably linked to a promoter, such as a constitutivepromoter (e.g., a cauliflower mosaic virus 35S promoter), or a cellspecific or inducible promoter. In particular embodiments, thepolynucleotide is introduced by microprojectile bombardment. Inparticular embodiments, the method further includes screening the plantcell after the introducing steps to determine whether the repairtemplate i.e. the gene of interest has been introduced. In particularembodiments, the methods include the step of regenerating a plant fromthe plant cell. In further embodiments, the methods include crossbreeding the plant to obtain a genetically desired plant lineage.Examples of foreign genes encoding a trait of interest are listed below.

b) Editing of Endogenous Genes to Confer an Agricultural Trait ofInterest

The invention provides methods of genome editing or modifying sequencesassociated with or at a target locus of interest wherein the methodcomprises introducing a Cpf1 effector protein complex into a plant cell,whereby the Cpf1 complex modifies the expression of an endogenous geneof the plant. This can be achieved in different ways, In particularembodiments, the elimination of expression of an endogenous gene isdesirable and the Cpf1 CRISPR complex is used to target and cleave anendogenous gene so as to modify gene expression. In these embodiments,the methods provided herein include (a) introducing into the plant cella Cpf1 CRISPR complex comprising a guide RNA, comprising a direct repeatand a guide sequence, wherein the guide sequence hybrdizes to a targetsequence within a gene of interest in the genome of the plant cell; and(b) introducing into the cell a Cpf1 effector protein, which uponbinding to the guide RNA comprises a guide sequence that is hybridizedto the target sequence, ensures a double strand break at or near thesequence to which the guide sequence is targeted; In particularembodiments, the step of introducing can include delivering to the plantcell one or more polynucleotides encoding Cpf1 effector protein and theguide RNA.

In particular embodiments, the polynucleotides are delivered into thecell by a DNA virus (e.g., a geminivirus) or an RNA virus (e.g., atobravirus). In particular embodiments, the introducing steps includedelivering to the plant cell a T-DNA containing one or morepolynucleotide sequences encoding the Cpf1 effector protein and theguide RNA, where the delivering is via Agrobacterium. The polynucleotidesequence encoding the components of the Cpf1 CRISPR system can beoperably linked to a promoter, such as a constitutive promoter (e.g., acauliflower mosaic virus 35S promoter), or a cell specific or induciblepromoter. In particular embodiments, the polynucleotide is introduced bymicroprojectile bombardment. In particular embodiments, the methodfurther includes screening the plant cell after the introducing steps todetermine whether the expression of the gene of interest has beenmodified. In particular embodiments, the methods include the step ofregenerating a plant from the plant cell. In further embodiments, themethods include cross breeding the plant to obtain a genetically desiredplant lineage.

In particular embodiments of the methods described above, diseaseresistant crops are obtained by targeted mutation of diseasesusceptibility genes or genes encoding negative regulators (e.g. Mlogene) of plant defense genes. In a particular embodiment,herbicide-tolerant crops are generated by targeted substitution ofspecific nucleotides in plant genes such as those encoding acetolactatesynthase (ALS) and protoporphyrinogen oxidase (PPO). In particularembodiments drought and salt tolerant crops by targeted mutation ofgenes encoding negative regulators of abiotic stress tolerance, lowamylose grains by targeted mutation of Waxy gene, rice or other grainswith reduced rancidity by targeted mutation of major lipase genes inaleurone layer, etc. In particular embodiments. A more extensive list ofendogenous genes encoding a traits of interest are listed below.

c) Modulating of Endogenous Genes by the Cpf1 CRISPR System to Confer anAgricultural Trait of Interest

Also provided herein are methods for modulating (i.e. activating orrepressing) endogenous gene expression using the Cpf1 protein providedherein. Such methods make use of distinct RNA sequence(s) which aretargeted to the plant genome by the Cpf1 complex. More particularly thedistinct RNA sequence(s) bind to two or more adaptor proteins (e.g.aptamers) whereby each adaptor protein is associated with one or morefunctional domains and wherein at least one of the one or morefunctional domains associated with the adaptor protein have one or moreactivities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,DNA integration activity RNA cleavage activity, DNA cleavage activity ornucleic acid binding activity; The functional domains are used tomodulate expression of an endogenous plant gene so as to obtain thedesired trait. Typically, in these embodiments, the Cpf1 effectorprotein has one or more mutations such that it has no more than 5% ofthe nuclease activity of the Cpf1 effector protein not having the atleast one mutation.

In particular embodiments, the methods provided herein include the stepsof (a) introducing into the cell a Cpf1 CRISPR complex comprising aguide RNA, comprising a direct repeat and a guide sequence, wherein theguide sequence hybrdizes to a target sequence that is endogenous to theplant cell; (b) introducing into the plant cell a Cpf1 effector moleculewhich complexes with the guide RNA when the guide sequence hybridizes tothe target sequence; and wherein either the guide RNA is modified tocomprise a distinct RNA sequence (aptamer) binding to a functionaldomain and/or the Cpf1 effector protein is modified in that it is linkedto a functional domain. In particular embodiments, the step ofintroducing can include delivering to the plant cell one or morepolynucleotides encoding the (modified) Cpf1 effector protein and the(modified) guide RNA. The details the components of the Cpf1 CRISPRsystem for use in these methods are described elsewhere herein.

In particular embodiments, the polynucleotides are delivered into thecell by a DNA virus (e.g., a geminivirus) or an RNA virus (e.g., atobravirus). In particular embodiments, the introducing steps includedelivering to the plant cell a T-DNA containing one or morepolynucleotide sequences encoding the Cpf1 effector protein and theguide RNA, where the delivering is via Agrobacterium. The nucleic acidsequence encoding the one or more components of the Cpf1 CRISPR systemcan be operably linked to a promoter, such as a constitutive promoter(e.g., a cauliflower mosaic virus 35S promoter), or a cell specific orinducible promoter. In particular embodiments, the polynucleotide isintroduced by microprojectile bombardment. In particular embodiments,the method further includes screening the plant cell after theintroducing steps to determine whether the expression of the gene ofinterest has been modified. In particular embodiments, the methodsinclude the step of regenerating a plant from the plant cell. In furtherembodiments, the methods include cross breeding the plant to obtain agenetically desired plant lineage. A more extensive list of endogenousgenes encoding a traits of interest are listed below.

Use of Cpf1 to Modify Polyploid Plants

Many plants are polyploid, which means they carry duplicate copies oftheir genomes—sometimes as many as six, as in wheat. The methodsaccording to the present invention, which make use of the Cpf1 CRISPReffector protein can be “multiplexed” to affect all copies of a gene, orto target dozens of genes at once. For instance, in particularembodiments, the methods of the present invention are used tosimultaneously ensure a loss of function mutation in different genesresponsible for suppressing defences against a disease. In particularembodiments, the methods of the present invention are used tosimultaneously suppress the expression of the TaMLO-Al, TaMLO-Bl andTaMLO-Dl nucleic acid sequence in a wheat plant cell and regenerating awheat plant therefrom, in order to ensure that the wheat plant isresistant to powdery mildew (see also WO2015109752).

Exemplary Genes Conferring Agronomic Traits

As described herein above, in particular embodiments, the inventionencompasses the use of the Cpf1 CRISPR system as described herein forthe insertion of a DNA of interest, including one or more plantexpressible gene(s). In further particular embodiments, the inventionencompasses methods and tools using the Cpf1 system as described hereinfor partial or complete deletion of one or more plant expressed gene(s).In other further particular embodiments, the invention encompassesmethods and tools using the Cpf1 system as described herein to ensuremodification of one or more plant-expressed genes by mutation,substitution, insertion of one of more nucleotides. In other particularembodiments, the invention encompasses the use of Cpf1 CRISPR system asdescribed herein to ensure modification of expression of one or moreplant-expressed genes by specific modification of one or more of theregulatory elements directing expression of said genes.

In particular embodiments, the invention encompasses methods whichinvolve the introduction of exogenous genes and/or the targeting ofendogenous genes and their regulatory elements, such as listed below:

1. Genes that Confer Resistance to Pests or Diseases:

-   -   Plant disease resistance genes. A plant can be transformed with        cloned resistance genes to engineer plants that are resistant to        specific pathogen strains. See, e.g., Jones et al., Science        266:789 (1994) (cloning of the tomato Cf-9 gene for resistance        to Cladosporium fulvum); Martin et al., Science 262:1432 (1993)        (tomato Pto gene for resistance to Pseudomonas syringae pv.        tomato encodes a protein kinase); Mindrinos et al., Cell        78:1089 (1994) (Arabidopsmay be RSP2 gene for resistance to        Pseudomonas syringae).    -   Genes conferring resistance to a pest, such as soybean cyst        nematode. See e.g, PCT Application WO 96/30517; PCT Application        WO 93/19181.    -   Bacillus thuringiensis proteins see, e.g., Geiser et al., Gene        48:109 (1986).    -   Lectins, see, for example, Van Damme et al., Plant Molec, Biol.        24:25 (1994.    -   Vitamin-binding protein, such as avidin, see PCT application        US93/06487, teaching the use of avidin and avidin homologues as        larvicides against insect pests.    -   Enzyme inhibitors such as protease or proteinase inhibitors or        amylase inhibitors. See, e.g., Abe et al., J. Biol. Chem.        262:16793 (1987), Huub et al., Plant Molec. Biol. 21:985        (1993)), Sumitani et al., Biosci. Biotech. Biochem.        57:1243 (1993) and U.S. Pat. No. 5,494,813.    -   Insect-specific hormones or pheromones such as ecdysteroid or        juvenile hormone, a variant thereof, a mimetic based thereon, or        an antagonist or agonist thereof. See, for example Hammock et        al., Nature 344:458 (1990).    -   Insect-specific peptides or neuropeptides which, upon        expression, disrupts the physiology of the affected pest. For        example Regan, J. Biol. Chem. 269:9 (1994) and Pratt et al.,        Biochem. Biophys. Res. Comm. 163:1243 (1989). See also U.S. Pat.        No. 5,266,317.    -   Insect-specific venom produced in nature by a snake, a wasp, or        any other organism. For example, see Pang et al., Gene 116: 165        (1992).    -   Enzymes responsible for a hyperaccumulation of a monoterpene, a        sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid        derivative or another nonprotein molecule with insecticidal        activity.    -   Enzymes involved in the modification, including the        post-translational modification, of a biologically active        molecule; for example, a glycolytic enzyme, a proteolytic        enzyme, a lipolytic enzyme, a nuclease, a cyclase, a        transaminase, an esterase, a hydrolase, a phosphatase, a kinase,        a phosphorylase, a polymerase, an elastase, a chitinase and a        glucanase, whether natural or synthetic. See PCT application        WO93/02197, Kramer et al., Insect Biochem, Molec. Biol.        23:691 (1993) and Kawalieck et al., Plant Molec. Biol. 21:673        (1993).    -   Molecules that stimulates signal transduction. For example, see        Botella et al., Plant Molec, Biol. 24:757 (1994), and Griess et        al., Plant Physiol. 104:1467 (1994).    -   Viral-invasive proteins or a complex toxin derived therefrom.        See Beachy et al., Ann. rev. Phytopathol. 28:451 (1990).    -   Developmental-arrestive proteins produced in nature by a        pathogen or a parasite. See Lamb et al., Bio/Technology        10:1436 (1992) and Toubart et al., Plant J. 2:367 (1992).    -   A developmental-arrestive protein produced in nature by a plant.        For example, Logemann et al., Bio/Technology 10:305 (1992).    -   In plants, pathogens are often host-specific. For example, some        Fusarium species will causes tomato wilt but attacks only        tomato, and other Fusarium species attack only wheat. Plants        have existing and induced defenses to resist most pathogens.        Mutations and recombination events across plant generations lead        to genetic variability that gives rise to susceptibility,        especially as pathogens reproduce with more frequency than        plants. In plants there can be non-host resistance, e.g., the        host and pathogen are incompatible or there can be partial        resistance against all races of a pathogen, typically controlled        by many genes and/or also complete resistance to some races of a        pathogen but not to other races, Such resistance is typically        controlled by a few genes. Using methods and components of the        CRISP-cpf1 system, a new tool now exists to induce specific        mutations in anticipation hereon. Accordingly, one can analyze        the genome of sources of resistance genes, and in plants having        desired characteristics or traits, use the method and components        of the Cpf1 CRISPR system to induce the rise of resistance        genes. The present systems can do so with more precision than        previous mutagenic agents and hence accelerate and improve plant        breeding programs.

2. Genes Involved in Plant Diseases, Such as Those Listed in WO2013046247:

-   -   Rice diseases: Magnaporthe grisea, Cochliobolus miyabeanus,        Rhizoctonia solani, Gibberella fujikuroi; Wheat diseases:        Erysiphe graminis, Fusarium graminearum, F. avenaceum, F.        culmorum, Microdochium nivale, Puccinia striiformis, P.        graminis, P. recondita, Micronectriella nivale, Typhula sp.,        Ustilago tritici, Tilletia caries, Pseudocercosporella        herpotrichoides, Mycosphaerella graminicola, Stagonospora        nodorum, Pyrenophora tritici-repentis; Barley diseases: Erysiphe        graminis, Fusarium graminearum, F. avenaceum, F. culmorum,        Microdochium nivale, Puccinia striiformis, P. graminis, P.        hordei, Ustilago nuda, Rhynchosporium secalis, Pyrenophora        teres, Cochliobolus sativus, Pyrenophora graminea, Rhizoctonia        solani; Maize diseases: Ustilago maydis, Cochliobolus        heterostrophus, Gloeocercospora sorghi, Puccinia polysora,        Cercospora zeae-maydis, Rhizoctonia solani;    -   Citrus diseases: Diaporthe citri, Elsinoe fawcetti, Penicillium        digitatum, P. italicum, Phytophthora parasitica, Phytophthora        citrophthora; Apple diseases: Monilinia mali, Valsa        ceratosperma, Podosphaera leucotricha, Alternaria alternata        apple pathotype, Venturia inaequalis, Colletotrichum acutatum,        Phytophtora cactorum;    -   Pear diseases: Venturia nashicola, V. pirina, Alternaria        alternata Japanese pear pathotype, Gymnosporangium haraeanium,        Phytophtora cactorum;    -   Peach diseases: Monilinia fructicola, Cladosporium carpophilum,        Phomopsis sp.;    -   Grape diseases: Elsinoe ampelina, Glomerella cingulata, Uninula        necator, Phakopsora ampelopsidis, Guignardia bidwelii,        Plasmopara viticola;    -   Persimmon diseases: Gloesporium kaki, Cercospora kaki,        Mycosphaerela nawae;    -   Gourd diseases: Colletotrichum lagenarium, Sphaerotheca        fuliginea, Mycosphaerella melonis, Fusarium oxysporum,        Pseudoperonospora cubensis, Phytophthora sp., Pythium sp.;    -   Tomato diseases: Alternaria solani, Cladosporium fulvum,        Phytophthora infestans,    -   Eggplant diseases: Phomopsis vexans, Erysiphe cichoracearum;        Brassicaceous vegetable diseases: Alternaria japonica,        Cercosporella brassicae, Plasmodiophora brassicae, Peronospora        parasitica;    -   Welsh onion diseases: Puccinia allii, Peronospora destructor;    -   Soybean diseases: Cercospora kikuchii, Elsinoe glycines,        Diaporthe phaseolorum var. sojae, Septoria glycines, Cercospora        sojina, Phakopsora pachyrhizi, Phytophthora sojae, Rhizoctonia        solani, Corynespora casiicola, Sclerotinia sclerotiorum,    -   Kidney bean diseases: Colletrichum lindemthianum,    -   Peanut diseases: Cercospora personata, Cercospora arachidicola,        Sclerotium rolfsii;    -   Pea diseases pea: Erysiphe pisi;    -   Potato diseases: Alternaria solani, Phytophthora infestans,        Phytophthora erythroseptica, Spongospora subterranean, f. sp.        Subterranean;    -   Strawberry diseases: Sphaerotheca humuli, Glomerella cingulata;    -   Tea diseases: Exobasidium reticulatum, Elsinoe leucospila,        Pestaotiopsis sp., Colletotrichum theae-sinensis;    -   Tobacco diseases: Alternaria longipes, Erysiphe cichoracearum,        Colletotrichum tabacum, Peronospora tabacina, Phytophthora        nicotianae;    -   Rapeseed diseases: Sclerotinia sclerotiorum, Rhizoctonia solani;    -   Cotton diseases: Rhizoctonia solani:    -   Beet diseases: Cercospora beticola, Thanatephorus cucumeris,        Thanatephorus cucumeris, Aphanomyces cochlioides;    -   Rose diseases: Diplocarpon rosae, Sphaerotheca pannosa,        Peronospora sparsa;    -   Diseases of chrysanthemum and asteraceae: Bremia lactuca,        Septoria chrysanthemi-indici, Puccinia horiana;    -   Diseases of various plants: Pythium aphanidermatum, Pythium        debarianum, Pythium graminicola, Pythium irregulare, Pythium        ultimum, Botrytis cinerea, Sclerotinia sclerotiorum;    -   Radish diseases: Alternaria brassicicola;    -   Zoysia diseases: Sclerotinia homeocarpa, Rhizoctonia    -   Banana diseases: Mycosphaerella fijiensis, Mycosphaerella        musicola;    -   Sunflower diseases: Plasmopara halstedii;    -   Seed diseases or diseases in the initial stage of growth of        various plants caused by Aspergillus spp., Penicillium spp.,        Fusarium spp., Gibberella spp., Tricoderma spp., Thielaviopsis        spp., Rhizopus spp., Mucor spp., Corticium spp., Rhoma spp.,        Rhizoctonia spp., Diplodia. spp., or the like;    -   Virus diseases of various plants mediated by Polymixa spp.,        Olpidium spp., or the like.

3. Examples of Genes that Confer Resistance to Herbicides:

-   -   Resistance to herbicides that inhibit the growing point or        meristem, such as an imidazolinone or a sulfonylurea, for        example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al.,        Theor. Appl. Genet. 80:449 (1990), respectively.    -   Glyphosate tolerance (resistance conferred by, e.g., mutant        5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes, aroA        genes and glyphosate acetyl transferase (GAT) genes,        respectively), or resistance to other phosphono compounds such        as by glufosinate (phosphinothricin acetyl transferase (PAT)        genes from Streptomyces species, including Streptomyces        hygroscopicus and Streptomyces viridichromogenes), and to        pyridinoxy or phenoxy proprionic acids and cyclohexones by        ACCase inhibitor-encoding genes. See, for example, U.S. Pat.        Nos. 4,940,835 and 6,248,876, 4,769,061, EP No. 0 333 033 and        U.S. Pat. No. 4,975,374. See also EP No. 0242246, DeGreef et        al., Bio/Technology 7:61 (1989), Marshall et al., Theor. Appl.        Genet, 83:435 (1992), WO 2005012515 to Castle et. al. and WO        2005107437.    -   Resistance to herbicides that inhibit photosynthesis, such as a        triazine (psbA and gs+ genes) or a benzonitrile (nitrilase        gene), and glutathione S-transferase in Przibila et al., Plant        Cell 3:169 (1991), U.S. Pat. No. 4,810,648, and Hayes et al.,        Biochem. J. 285: 173 (1992).    -   Genes encoding Enzymes detoxifying the herbicide or a mutant        glutamine synthase enzyme that is resistant to inhibition, e.g.        n U.S. patent application Ser. No. 11/760,602. Or a detoxifying        enzyme is an enzyme encoding a phosphinothricin        acetyltransferase (such as the bar or pat protein from        Streptomyces species). Phosphinothricin acetyltransferases are        for example described in U.S. Pat. Nos. 5,561,236; 5,648,477;        5,646,024; 5,273,894; 5,637,489; 5,276,268; 5,739,082; 5,908,810        and 7,112,665. Hydroxyphenylpyruyatedioxygenases (RPM)        inhibitors, ie naturally occurring HPPD resistant enzymes, or        genes encoding a mutated or chimeric HPPD enzyme as described in        WO 96/38567, WO 99/24585, and WO 99/24586, WO 2009/144079, WO        2002/046387, or U.S. Pat. No. 6,768,044.

4. Examples of Genes Involved in Abiotic Stress Tolerance:

-   -   Transgene capable of reducing the expression and/or the activity        of poly(ADP-ribose) polymerase (PARP) gene in the plant cells or        plants as described in WO 00/04173 or, WO/2006/045633.    -   Transgenes capable of reducing the expression and/or the        activity of the PARG encoding genes of the plants or plants        cells, as described e.g. in WO 2004/090140,    -   Transgenes coding for a plant-functional enzyme of the        nicotineamide adenine dinucleotide salvage synthesis pathway        including nicotinamidase, nicotinate phosphoribosyltransferase,        nicotinic acid mononucleotide adenyl transferase, nicotinamide        adenine dinucleotide synthetase or nicotine amide        phosphorybosyltransferase as described e.g. in EP 04077624.7, WO        2006/133827, PCT/EP07/002,433, EP 1999263, or WO 2007/107326.    -   Enzymes involved in carbohydrate biosynthesis include those        described in e.g. EP 0571427, WO 95/04826, EP 0719338, WO        96/15248, WO 96/19581, WO 96/27674, WO 97/11188, WO 97/26362, WO        97/32985, WO 97/42328, WO 97/44472, WO 97/45545, WO 98/27212, WO        98/40503, WO99/58688, WO 99/58690, WO 99/58654, WO 00/08184, WO        00/08185, WO 00/08175, WO 00/28052, WO 00/77229, WO 01/12782, WO        01/12826, WO 02/101059, WO 03/071860, WO 2004/056999, WO        2005/030942, WO 2005/030941, WO 2005/095632, WO 2005/095617, WO        2005/095619, WO 2005/095618, WO 2005/123927, WO 2006/018319, WO        2006/103107, WO 2006/108702, WO 2007/009823, WO 00/22140, WO        2006/063862, WO 2006/072603, WO 02/034923, EP 06090134.5, EP        06090228.5, EP 06090227.7, EP 07090007.1, EP 07090009.7, WO        01/14569, WO 02/79410, WO 03/33540, WO 2004/078983, WO 01/19975,        WO 95/26407 WO 96/34968, WO 98/20145, WO 99/12950, WO 99/66050,        WO 99/53072, U.S. Pat. No. 6,734,341, WO 00/11192, WO 98/22604,        WO 98/32326, WO 01/98509, WO 01/98509, WO 2005/002359, U.S. Pat.        Nos. 5,824,790, 6,013,861, WO 94/04693, WO 94/09144, WO 94R1520,        WO 95/35026 or WO 97/20936 or enzymes involved in the production        of polyfructose, especially of the inulin and levan-type, as        disclosed in EP 0663956, WO 96/01904, WO 96/21023, WO 98/39460,        and WO 99/24593, the production of alpha-1,4-glucans as        disclosed in WO 95/31553, US 2002031826, U.S. Pat. Nos.        6,284,479, 5,712,107, WO 97/47806, WO 97/47807, WO 97/47808 and        WO 00/14249, the production of alpha-1,6 branched        alpha-1,4-glucans, as disclosed in WO 00/73422, the production        of alternan, as disclosed in e.g. WO 00/47727, WO 00/73422, EP        06077301.7, U.S. Pat. No. 5,908,975 and EP 0728213, the        production of hyaluronan, as for example disclosed in WO        2006/032538, WO 2007/039314, WO 2007/039315, WO 2007/039316, JP        2006304779, and WO 2005/012529.    -   Genes that improve drought resistance. For example, WO        2013122472 discloses that the absence or reduced level of        functional Ubiquitin Protein Ligase protein (UPL) protein, more        specifically, UPL3, leads to a decreased need for water or        improved resistance to drought of said plant. Other examples of        transgenic plants with increased drought tolerance are disclosed        in, for example, US 2009/0144850, US 2007/0266453, and WO        2002/083911. US2009/0144850 describes a plant displaying a        drought tolerance phenotype due to altered expression of a DR02        nucleic acid. US 2007/0266453 describes a plant displaying a        drought tolerance phenotype due to altered expression of a DR03        nucleic acid and WO 2002/08391 1 describes a plant having an        increased tolerance to drought stress due to a reduced activity        of an ABC transporter which is expressed in guard cells. Another        example is the work by Kasuga and co-authors (1999), who        describe that overexpression of cDNA encoding DREB1 A in        transgenic plants activated the expression of many stress        tolerance genes under normal growing conditions and resulted in        improved tolerance to drought, salt loading, and freezing.        However, the expression of DREB1A also resulted in severe growth        retardation under normal growing conditions (Kasuga (1999) Nat        Biotechnol 17(3) 287-291).

In further particular embodiments, crop plants can be improved byinfluencing specific plant traits. For example, by developingpesticide-resistant plants, improving disease resistance in plants,improving plant insect and nematode resistance, improving plantresistance against parasitic weeds, improving plant drought tolerance,improving plant nutritional value, improving plant stress tolerance,avoiding self-pollination, plant forage digestibility biomass, grainyield etc. A few specific non-limiting examples are providedhereinbelow.

In addition to targeted mutation of single genes, Cpf1 CRISPR complexescan be designed to allow targeted mutation of multiple genes, deletionof chromosomal fragment, site-specific integration of transgene,site-directed mutagenesis in vivo, and precise gene replacement orallele swapping in plants. Therefore, the methods described herein havebroad applications in gene discovery and validation, mutational andcisgenic breeding, and hybrid breeding. These applications facilitatethe production of a new generation of genetically modified crops withvarious improved agronomic traits such as herbicide resistance, diseaseresistance, abiotic stress tolerance, high yield, and superior quality.

Use of Cpf1 Gene to Create Male Sterile Plants

Hybrid plants typically have advantageous agronomic traits compared toinbred plants. However, for self-pollinating plants, the generation ofhybrids can be challenging. In different plant types, genes have beenidentified which are important for plant fertility, more particularlymale fertility. For instance, in maize, at least two genes have beenidentified which are important in fertility (Amitabh MohantyInternational Conference on New Plant. Breeding Molecular TechnologiesTechnology Development And Regulation, Oct. 9-10, 2014, Jaipur, India;Svitashev et al. Plant. Physiol. 2015 October, 169(2):931-45; Djukanovicet al. Plant J. 2013 December; 76(5):888-99). The methods providedherein can be used to target genes required for male fertility so as togenerate male sterile plants which can easily be crossed to generatehybrids. In particular embodiments, the Cpf1 CRISPR system providedherein is used for targeted mutagenesis of the cytochrome P450-like gene(MS26) or the meganuclease gene (MS45) thereby conferring male sterilityto the maize plant. Maize plants which are as such genetically alteredcan be used in hybrid breeding programs.

Increasing the Fertility Stage in Plants

In particular embodiments, the methods provided herein are used toprolong the fertility stage of a plant such as of a rice plant. Forinstance, a rice fertility stage gene such as Ehd3 can be targeted inorder to generate a mutation in the gene and plantlets can be selectedfor a prolonged regeneration plant fertility stage (as described in CN104004782)

Use of Cpf1 to Generate Genetic Variation in a Crop of Interest

The availability of wild germplasm and genetic variations in crop plantsis the key to crop improvement programs, but the available diversity ingermplasms from crop plants is limited. The present invention envisagesmethods for generating a diversity of genetic variations in a germplasmof interest. In this application of the Cpf1. CRISPR system a library ofguide RNAs targeting different locations in the plant genome is providedand is introduced into plant cells together with the Cpf1 effectorprotein. In this way a collection of genome-scale point mutations andgene knock-outs can be generated. In particular embodiments, the methodscomprise generating a plant part or plant from the cells so obtained andscreening the cells for a trait of interest. The target genes caninclude both coding and non-coding regions. In particular embodiments,the trait is stress tolerance and the method is a method for thegeneration of stress-tolerant crop varieties

Use of Cpf1 to Affect Fruit-Ripening

Ripening is a normal phase in the maturation process of fruits andvegetables. Only a few days after it starts it renders a fruit orvegetable inedible. This process brings significant losses to bothfarmers and consumers. In particular embodiments, the methods of thepresent invention are used to reduce ethylene production. This isensured by ensuring one or more of the following: a. Suppression of ACCsynthase gene expression. ACC (1-aminocyclopropane-1-carboxylic acid)synthase is the enzyme responsible for the conversion ofS-adenosylmethionine (SAM) to ACC; the second to the last step inethylene biosynthesis. Enzyme expression is hindered when an antisense(“mirror-image”) or truncated copy of the synthase gene is inserted intothe plant's genome; b. Insertion of the ACC deaminase gene. The genecoding for the enzyme is obtained from Pseudomonas chlororaphis, acommon nonpathogenic soil bacterium. It converts ACC to a differentcompound thereby reducing the amount of ACC available for ethyleneproduction; c. Insertion of the SAM hydrolase gene. This approach issimilar to ACC deaminase wherein ethylene production is hindered whenthe amount of its precursor metabolite is reduced; in this case SAM isconverted to homoserine. The gene coding for the enzyme is obtained fromE. coli T3 bacteriophage and d. Suppression of ACC oxidase geneexpression. ACC oxidase is the enzyme which catalyzes the oxidation ofACC to ethylene, the last step in the ethylene biosynthetic pathway.Using the methods described herein, down regulation of the ACC oxidasegene results in the suppression of ethylene production, thereby delayingfruit ripening. In particular embodiments, additionally or alternativelyto the modifications described above, the methods described herein areused to modify ethylene receptors, so as to interfere with ethylenesignals obtained by the fruit. In particular embodiments, expression ofthe ETR1 gene, encoding an ethylene binding protein is modified, moreparticularly suppressed. In particular embodiments, additionally oralternatively to the modifications described above, the methodsdescribed herein are used to modify expression of the gene encodingPolygalacturonase (PG), which is the enzyme responsible for thebreakdown of pectin, the substance that maintains the integrity of plantcell walls. Pectin breakdown occurs at the start of the ripening processresulting in the softening of the fruit. Accordingly, in particularembodiments, the methods described herein are used to introduce amutation in the PG gene or to suppress activation of the PG gene inorder to reduce the amount of PG enzyme produced thereby delaying pectindegradation.

Thus in particular embodiments, the methods comprise the use of the Cpf1CRISPR system to ensure one or more modifications of the genome of aplant cell such as described above, and regenerating a plant therefrom.In particular embodiments, the plant is a tomato plant.

Increasing Storage Life of Plants

In particular embodiments, the methods of the present invention are usedto modify genes involved in the production of compounds which affectstorage life of the plant or plant part. More particularly, themodification is in a gene that prevents the accumulation of reducingsugars in potato tubers. Upon high-temperature processing, thesereducing sugars react with free amino acids, resulting in brown,bitter-tasting products and elevated levels of acrylamide, which is apotential carcinogen. In particular embodiments, the methods providedherein are used to reduce or inhibit expression of the vacuolarinvertase gene (VInv), which encodes a protein that breaks down sucroseto glucose and fructose (Clasen et al. DOI: 10.1111/pbi.12370).

The Use of the Cpf1 CRISPR System to Ensure a Value Added Trait

In particular embodiments the Cpf1 CRISPR system is used to producenutritionally improved agricultural crops. In particular embodiments,the methods provided herein are adapted to generate “functional foods”,i.e. a modified food or food ingredient that may provide a healthbenefit beyond the traditional nutrients it contains and or“nutraceutical”, i.e. substances that may be considered a food or partof a food and provides health benefits, including the prevention andtreatment of disease. In particular embodiments, the nutraceutical isuseful in the prevention and/or treatment of one or more of cancer,diabetes, cardiovascular disease, and hypertension.

Examples of nutritionally improved crops include (Newell-McGloughlin,Plant Physiology, July 2008, Vol. 147, pp. 939-953):

-   -   modified protein quality, content and/or amino acid composition,        such as have been described for Bahiagrass (Luciani et al. 2005,        Florida Genetics Conference Poster), Canola (Roesler et al.,        1997, Plant Physiol 113 75-81), Maize (Cromwell et al, 1967,        1969 J Anim Sci 26 1325-1331, O'Quin et al. 2000 J Anim Sci 78        2144-2149, Yang et al. 2002, Transgenic Res 11 11-20, Young et        al. 2004, Plant J 38 910-922), Potato (Yu J and Ao, 1997 Acta        Bot Sin 39 329-334; Chakraborty et al. 2000, Proc Natl Acad Sci        USA 97 3724-3729; Li et al. 2001) Chin Sci Bull 46 482-484, Rice        (Katsube et al. 1999, Plant Physiol 120 1063-1074), Soybean        (Dinkins et al. 2001, Rapp 2002, In Vitro Cell Dev Biol Plant 37        742-747), Sweet Potato (Egnin and Prakash 1997, In Vitro Cell        Dev Biol 33 52A).    -   essential amino acid content, such as has been described for        Canola (Falco et al. 1995, Bio/Technology 13 577-582), Lupin        (White et al. 2001, J Sci Food Agric 81 147-154), Maize (Lai and        Messing, 2002, Agbios 2008 GM crop database (Mar. 11, 2008)),        Potato (Zeh et al. 2001, Plant Physiol 127 792-802), Sorghum        (Zhao et al. 2003, Kluwer Academic Publishers, Dordrecht, The        Netherlands, pp 413-416), Soybean (Falco et al. 1995        Bio/Technology 13 577-582; Galili et al. 2002 Crit Rev Plant Sci        21 167-204).    -   Oils and Fatty acids such as for Canola (Dehesh et al. (1996)        Plant J 9 167-172 [PubMed]; Del Vecchio (1996) INFORM        International News on Fats, Oils and Related Materials 7        230-243; Roesler et al. (1997) Plant Physiol 113 75-81 [PMC free        article] [PubMed]; Froman and Ursin (2002, 2003) Abstracts of        Papers of the American Chemical Society 223 U35; James et        al. (2003) Am J Clin Nutr 77 1140-1145 [PubMed]; Agbios (2008,        above); coton (Chapman et al. (2001). J Am Oil Chem Soc 78        941-947; Liu et al. (2002) J Am Coll Nutr 21 205S-211S [PubMed];        O'Neill (2007) Australian Life Scientist.        http://www.biotechnews.com.au/index.php/id;866694817;fp;4;fpid;2        (Jun. 17, 2008), Linseed (Abbadi et al., 2004, Plant Cell 16:        2734-2748), Maize (Young et al., 2004, Plant J 38 910-922), oil        palm (Jalani et al. 1997, J Am Oil Chem Soc 74 1451-1455;        Parveez, 2003, AgBiotechNet 113 1-8), Rice (Anai et al., 2003,        Plant Cell Rep 21 988-992), Soybean (Reddy and Thomas, 1996, Nat        Biotechnol 14 639-642; Kinney and Kwolton, 1998, Blackie        Academic and Professional, London, pp 193-213), Sunflower        (Arcadia, Biosciences 2008)    -   Carbohydrates, such as Fructans described for Chicory        (Smeekens (1997) Trends Plant Sci 2 286-287, Sprenger et        al. (1997) FEBS Lett 400 355-358, Sévenier et al. (1998) Nat        Biotechnol 16 843-846), Maize (Caimi et al. (1996) Plant Physiol        110 355-363), Potato (Hellwege et al., 1997 Plant J 12        1057-1065), Sugar Beet (Smeekens et al. 1997, above), Inulin,        such as described for Potato (Hellewege et al. 2000, Proc Natl        Acad Sci USA 97 8699-8704), Starch, such as described for Rice        (Schwall et al. (2000) Nat Biotechnol 18 551-554, Chiang et        al. (2005) Mol Breed 15 125-143),    -   Vitamins and carotenoids, such as described for Canola (Shintani        and DellaPenna (1998) Science 282 2098-2100), Maize (Rocheford        et al. (2002). J Am Coll Nutr 21 191S-198S, Cahoon et al. (2003)        Nat Biotechnol 21 1082-1087, Chen et al. (2003) Proc Natl Acad        Sci USA 100 3525-3530), Mustardseed (Shewmaker et al. (1999)        Plant J 20 401-412, Potato (Ducreux et al., 2005, J Exp Bot 56        81-89), Rice (Ye et al. (2000) Science 287 303-305, Strawberry        (Agius et al. (2003), Nat Biotechnol 21 177-181), Tomato (Rosati        et al. (2000) Plant J 24 413-419, Fraser et al. (2001) J Sci        Food Agric 81 822-827, Mehta et al. (2002) Nat Biotechnol 20        613-618, Diaz de la Garza et al. (2004) Proc Natl Acad Sci USA        101 13720-13725, Enfissi et al. (2005) Plant Biotechnol J 3        17-27, DellaPenna (2007) Proc Natl Acad Sci USA 104 3675-3676.    -   Functional secondary metabolites, such as described for Apple        (stilbenes, Szankowski et al. (2003) Plant Cell Rep 22:        141-149), Alfalfa (resveratrol, Hipskind and Paiva (2000) Mol        Plant Microbe Interact 13 551-562), Kiwi (resveratrol, Kobayashi        et al. (2000) Plant Cell Rep 19 904-910), Maize and Soybean        (flavonoids, Yu et al. (2000) Plant Physiol 124 781-794), Potato        (anthocyanin and alkaloid glycoside, Lukaszewicz et al. (2004) J        Agric Food Chem 52 1526-1533), Rice (flavonoids & resveratrol,        Stark-Lorenzen et al. (1997) Plant Cell Rep 16 668-673, Shin et        al. (2006) Plant Biotechnol J 4 303-315), Tomato (+resveratrol,        chlorogenic acid, flavonoids, stilbene; Rosati et al. (2000)        above, Muir et al. (2001) Nature 19 470-474, Niggeweg et        al. (2004) Nat Biotechnol 22 746-754, Giovinazzo et al. (2005)        Plant Biotechnol J 3 57-69), wheat (caffeic and ferulic acids,        resveratrol; United Press International (2002)); and    -   Mineral availabilities such as described for Alfalfa (phytase,        Austin-Phillips et al. (1999)        http://www.molecularfarming.com/nonmedical.html), Lettuse (iron,        Goto et al. (2000) Theor Appl Genet 100 658-664), Rice (iron,        Lucca et al. (2002) J Am Coll Nutr 21 184S-190S), Maize, Soybean        and wheate (phytase, Drakakaki et al. (2005) Plant Mol Biol 59        869-880, Denbow et al. (1998) Poult Sci 77 878-881,        Brinch-Pedersen et al. (2000) Mol Breed 6 195-206).

In particular embodiments, the value-added trait is related to theenvisaged health benefits of the compounds present in the plant. Forinstance, in particular embodiments, the value-added crop is obtained byapplying the methods of the invention to ensure the modification of orinduce/increase the synthesis of one or more of the following compounds:

-   -   Carotenoids, such as ix-Carotene present in carrots which        Neutralizes free radicals that may cause damage to cells or        β-Carotene present in various fruits and vegetables which        neutralizes free radicals    -   Lutein present in green vegetables which contributes to        maintenance of healthy vision    -   Lycopene present in tomato and tomato products, which is        believed to reduce the risk of prostate cancer    -   Zeaxanthin, present in citrus and maize, which contributes to        maintenance of healthy vision    -   Dietary fiber such as insoluble fiber present in wheat bran        which may reduce the risk of breast and/or colon cancer and        β-Glucan present in oat, soluble fiber present in Psylium and        whole cereal grains which may reduce the risk of cardiovascular        disease (CVD)    -   Fatty acids, such as ω-3 fatty acids which may reduce the risk        of CND and improve mental and visual functions, Conjugated        linoleic acid, which may improve body, composition, may decrease        risk of certain cancers and GLA which may reduce inflammation        risk of cancer and CVD, may improve body composition    -   Flavonoids such as Hydroxycinnamates, present in wheat which        have Antioxidant-like activities, may reduce risk of        degenerative diseases, flavonols, catechins and tannins present        in fruits and vegetables which neutralize free radicals and may        reduce risk of cancer    -   Glucosinolates, indoles, isothiocyanates, such as Sulforaphane,        present in Cruciferous vegetables (broccoli, kale), horseradish,        which neutralize free radicals, may reduce risk of cancer    -   Phenolics, such as stilbenes present in grape which May reduce        risk of degenerative diseases, heart disease, and cancer, may        have longevity effect and caffeic acid and ferulic acid present        in vegetables and citrus which have Antioxidant-like activities,        may reduce risk of degenerative diseases, heart disease, and eye        disease, and epicatechin present in cacao which has        Antioxidant-like activities, may reduce risk of degenerative        diseases and heart disease    -   Plant stanols/sterols present in maize, soy, wheat and wooden        oils which May reduce risk of coronary heart disease by lowering        blood cholesterol levels Fructans, inulins,        fructo-oligosaccharides present in Jerusalem artichoke, shallot,        onion powder which may improve gastrointestinal health    -   Saponins present in soybean, which may lower LDL cholesterol    -   Soybean protein present in soybean which may reduce risk of        heart disease    -   Phytoestrogens such as isoflavones present in soybean which May        reduce menopause symptoms, such as hot flashes, may reduce        osteoporosis and CVD and lignins present in flax, rye and        vegetables, which May protect against heart disease and some        cancers, may lower LDL cholesterol, total cholesterol.    -   Sulfides and thiols such as diallyl sulphide present in onion,        garlic, olive, leek and scallop and Allyl methyl trisulfide,        dithiolthiones present in cruciferous vegetables which may lower        LDL cholesterol, helps to maintain healthy immune system    -   Tannins, such as proanthocyanidins, present in cranberry, cocoa,        which may improve urinary tract health, may reduce risk of CVD        and high blood pressure    -   Etc.

In addition, the methods of the present invention also envisagemodifying protein/starch functionality, shelf life, taste/aesthetics,fiber quality, and allergen, antinutrient, and toxin reduction traits.

Accordingly, the invention encompasses methods for producing plants withnutritional added value, said methods comprising introducing into aplant cell a gene encoding an enzyme involved in the production of acomponent of added nutritional value using the Cpf1 CRISPR system asdescribed herein and regenerating a plant from said plant cell, saidplant characterized in an increase expression of said component of addednutritional value. In particular embodiments, the Cpf1 CRISPR system isused to modify the endogenous synthesis of these compounds indirectly,e.g. by modifying one or more transcription factors that controls themetabolism of this compound. Methods for introducing a gene of interestinto a plant cell and/or modifying an endogenous gene using the Cpf1CRISPR system are described herein above.

Some specific examples of modifications in plants that have beenmodified to confer value-added traits are: plants with modified fattyacid metabolism, for example, by transforming a plant with an antisensegene of stearyl-ACP desaturase to increase stearic acid content of theplant. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624(1992). Another example involves decreasing phytate content, for exampleby cloning and then reintroducing DNA associated with the single allelewhich may be responsible for maize mutants characterized by low levelsof phytic acid. See Raboy et al, Maydica 35:383 (1990).

Similarly, expression of the maize (Zea mays) Tfs C1 and R, whichregulate the production of flavonoids in maize aleurone layers under thecontrol of a strong promoter, resulted in a high accumulation rate ofanthocyanins in Arabidopsis (Arabidopsis thaliana), presumably byactivating the entire pathway (Bruce et al., 2000, Plant Cell 12:65-80).DellaPenna (Welsch et al., 2007 Annu Rev Plant Biol 57: 711-738) foundthat Tf RAP2.2 and its interacting partner SINAT2 increasedcarotenogenesis in Arabidopsis leaves. Expressing the Tf Dof1 inducedthe up-regulation of genes encoding enzymes for carbon skeletonproduction, a marked increase of amino acid content, and a reduction ofthe Glc level in transgenic Arabidopsis (Yanagisawa, 2004 Plant CellPhysiol 45: 386-391), and the DOF Tf AtDof1.1 (OBP2) up-regulated allsteps in the glucosinolate biosynthetic pathway in Arabidopsis (Skiryczet al., 2006 Plant J 47: 10-24).

Reducing Allergen in Plants

In particular embodiments the methods provided herein are used togenerate plants with a reduced level of allergens, making them safer forthe consumer. In particular embodiments, the methods comprise modifyingexpression of one or more genes responsible for the production of plantallergens. For instance, in particular embodiments, the methods comprisedown-regulating expression of a Lol p5 gene in a plant cell, such as aryegrass plant cell and regenerating a plant therefrom so as to reduceallergenicity of the pollen of said plant (Bhalla et al. 1999, Proc.Natl. Acad. Sci. USA Vol. 96: 11676-11680).

Peanut allergies and allergies to legumes generally are a real andserious health concern. The Cpf1 effector protein system of the presentinvention can be used to identify and then edit or silence genesencoding allergenic proteins of such legumes. Without limitation as tosuch genes and proteins, Nicolaou et al. identifies allergenic proteinsin peanuts, soybeans, lentils, peas, lupin, green beans, and mung beans.See, Nicolaou et al., Current Opinion in Allergy and Clinical Immunology2011; 11(3):222).

Screening Methods for Endogenous Genes of Interest

The methods provided herein further allow the identification of genes ofvalue encoding enzymes involved in the production of a component ofadded nutritional value or generally genes affecting agronomic traits ofinterest, across species, phyla, and plant kingdom. By selectivelytargeting e.g. genes encoding enzymes of metabolic pathways in plantsusing the Cpf1 CRISPR system as described herein, the genes responsiblefor certain nutritional aspects of a plant can be identified. Similarly,by selectively targeting genes which may affect a desirable agronomictrait, the relevant genes can be identified. Accordingly, the presentinvention encompasses screening methods for genes encoding enzymesinvolved in the production of compounds with a particular nutritionalvalue and/or agronomic traits.

Further Applications of the Cpf1 CRISPR System in Plants and Yeasts Useof Cpf1 CRISPR System in Biofuel Production

The term “biofuel” as used herein is an alternative fuel made from plantand plant-derived resources. Renewable biofuels can be extracted fromorganic matter whose energy has been obtained through a process ofcarbon fixation or are made through the use or conversion of biomass.This biomass can be used directly for biofuels or can be converted toconvenient energy containing substances by thermal conversion, chemicalconversion, and biochemical conversion. This biomass conversion canresult in fuel in solid, liquid, or gas form. There are two types ofbiofuels: bioethanol and biodiesel. Bioethanol is mainly produced by thesugar fermentation process of cellulose (starch), which is mostlyderived from maize and sugar cane. Biodiesel on the other hand is mainlyproduced from oil crops such as rapeseed, palm, and soybean. Biofuelsare used mainly for transportation.

Enhancing Plant Properties for Biofuel Production

In particular embodiments, the methods using the Cpf1 CRISPR system asdescribed herein are used to alter the properties of the cell wall inorder to facilitate access by key hydrolysing agents for a moreefficient release of sugars for fermentation. In particular embodiments,the biosynthesis of cellulose and/or lignin are modified. Cellulose isthe major component of the cell wall. The biosynthesis of cellulose andlignin are co-regulated. By reducing the proportion of lignin in a plantthe proportion of cellulose can be increased. In particular embodiments,the methods described herein are used to downregulate ligninbiosynthesis in the plant so as to increase fermentable carbohydrates.More particularly, the methods described herein are used to downregulateat least a first lignin biosynthesis gene selected from the groupconsisting of 4-coumarate 3-hydroxylase (C3H), phenylalanineammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamylalcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR),4-coumarate-CoA ligase (4CL), monolignol-lignin-specificglycosyltransferase, and aldehyde dehydrogenase (ALDH) as disclosed inWO 2008064289 A2.

In particular embodiments, the methods described herein are used toproduce plant mass that produces lower levels of acetic acid duringfermentation (see also WO 2010096488). More particularly, the methodsdisclosed herein are used to generate mutations in homologs to CaslL toreduce polysaccharide acetylation.

Modifying Yeast for Biofuel Production

In particular embodiments, the Cpf1 enzyme provided herein is used forbioethanol production by recombinant micro-organisms. For instance, Cpf1can be used to engineer micro-organisms, such as yeast, to generatebiofuel or biopolymers from fermentable sugars and optionally to be ableto degrade plant-derived lignocellulose derived from agricultural wasteas a source of fermentable sugars. More particularly, the inventionprovides methods whereby the Cpf1 CRISPR complex is used to introduceforeign genes required for biofuel production into micro-organismsand/or to modify endogenous genes why may interfere with the biofuelsynthesis. More particularly the methods involve introducing into amicro-organism such as a yeast one or more nucleotide sequence encodingenzymes involved in the conversion of pyruvate to ethanol or anotherproduct of interest. In particular embodiments the methods ensure theintroduction of one or more enzymes which allows the micro-organism todegrade cellulose, such as a cellulase. In yet further embodiments, theCpf1 CRISPR complex is used to modify endogenous metabolic pathwayswhich compete with the biofuel production pathway.

Accordingly, in more particular embodiments, the methods describedherein are used to modify a micro-organism as follows:

-   -   to introduce at least one heterologous nucleic acid or increase        expression of at least one endogenous nucleic acid encoding a        plant cell wall degrading enzyme, such that said micro-organism        is capable of expressing said nucleic acid and of producing and        secreting said plant cell wall degrading enzyme;    -   to introduce at least one heterologous nucleic acid or increase        expression of at least one endogenous nucleic acid encoding an        enzyme that converts pyruvate to acetaldehyde optionally        combined with at least one heterologous nucleic acid encoding an        enzyme that converts acetaldehyde to ethanol such that said host        cell is capable of expressing said nucleic acid; and/or    -   to modify at least one nucleic acid encoding for an enzyme in a        metabolic pathway in said host cell, wherein said pathway        produces a metabolite other than acetaldehyde from pyruvate or        ethanol from acetaldehyde, and wherein said modification results        in a reduced production of said metabolite, or to introduce at        least one nucleic acid encoding for an inhibitor of said enzyme.

Modifying Algae and Plants for Production of Vegetable Oils or Biofuels

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

According to particular embodiments of the invention, the Cpf1 CRISPRsystem is used to generate lipid-rich diatoms which are useful inbiofuel production.

In particular embodiments it is envisaged to specifically modify genesthat are involved in the modification of the quantity of lipids and/orthe quality of the lipids produced by the algal cell. Examples of genesencoding enzymes involved in the pathways of fatty acid synthesis canencode proteins having for instance acetyl-CoA carboxylase, fatty acidsynthase, 3-ketoacyl_acyl-carrier protein synthase III,glycerol-3-phosphate deshydrogenase (G3PDH), Enoyl-acyl carrier proteinreductase (Enoyl-ACP-reductase), glycerol-3-phosphate acyltransferase,lysophosphatidic acyl transferase or diacylglycerol acyltransferase,phospholipid: diacylglycerol acyltransferase, phoshatidate phosphatase,fatty acid thioesterase such as palmitoyl protein thioesterase, or malicenzyme activities. In further embodiments it is envisaged to generatediatoms that have increased lipid accumulation. This can be achieved bytargeting genes that decrease lipid catabolisation. Of particularinterest for use in the methods of the present invention are genesinvolved in the activation of both triacylglycerol and free fatty acids,as well as genes directly involved in β-oxidation of fatty acids, suchas acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA oxidaseactivity and phosphoglucomutase. The Cpf1 CRISPR system and methodsdescribed herein can be used to specifically activate such genes indiatoms as to increase their lipid content.

Organisms such as microalgae are widely used for synthetic biology.Stovicek et al. (Metab. Eng. Comm., 2015; 2:13 describes genome editingof industrial yeast, for example, Saccharomyces cerevisae, toefficiently produce robust strains for industrial production. Stovicekused a CRISPR-Cas9 system codon-optimized for yeast to simultaneouslydisrupt both alleles of an endogenous gene and knock in a heterologousgene. Cas9 and gRNA were expressed from genomic or episomal 2μ-basedvector locations. The authors also showed that gene disruptionefficiency could be improved by optimization of the levels of Cas9 andgRNA expression. Hlavová et al. (Biotechnol. Adv. 2015) discussesdevelopment of species or strains of microalgae using techniques such asCRISPR to target nuclear and chloroplast genes for insertionalmutagenesis and screening. The methods of Stovicek and Hlavová may beapplied to the Cpf1 effector protein system of the present invention.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the Cpf1 CRISPR system described herein can beapplied on Chlamydomonas species and other algae. In particularembodiments, Cpf1 and guide RNA are introduced in algae expressed usinga vector that expresses Cpf1 under the control of a constitutivepromoter such as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will bedelivered using a vector containing T7 promoter. Alternatively, Cpf1mRNA and in vitro transcribed guide RNA can be delivered to algal cells.Electroporation protocol follows standard recommended protocol from theGeneArt Chlamydomonas Engineering kit.

The Use of Cpf1 in the Generation of Micro-Organisms Capable of FattyAcid Production

In particular embodiments, the methods of the invention are used for thegeneration of genetically engineered micro-organisms capable of theproduction of fatty esters, such as fatty acid methyl esters (“FAME”)and fatty acid ethyl esters (“FAEE”),

Typically, host cells can be engineered to produce fatty esters from acarbon source, such as an alcohol, present in the medium, by expressionor overexpression of a gene encoding a thioesterase, a gene encoding anacyl-CoA synthase, and a gene encoding an ester synthase. Accordingly,the methods provided herein are used to modify a micro-organisms so asto overexpress or introduce a thioesterase gene, a gene encoding anacyl-CoA synthase, and a gene encoding an ester synthase. In particularembodiments, the thioesterase gene is selected from tesA, ‘tesA,tesB,fatB, fatB2,fatB3,fatA1, or fatA. In particular embodiments, thegene encoding an acyl-CoA synthase is selected from fadDJadK, BH3103,pfl-4354, EAV15023, fadD1, fadD2, RPC_4074,fadDD35, fadDD22, faa39, oran identified gene encoding an enzyme having the same properties. Inparticular embodiments, the gene encoding an ester synthase is a geneencoding a synthase/acyl-CoA:diacylglycerl acyltransferase fromSimmondsia chinensis, Acinetobacter sp. ADP, Alcanivorax borkumensis,Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, orAlkaligenes eutrophus, or a variant thereof. Additionally oralternatively, the methods provided herein are used to decreaseexpression in said micro-organism of at least one of a gene encoding anacyl-CoA dehydrogenase, a gene encoding an outer membrane proteinreceptor, and a gene encoding a transcriptional regulator of fatty acidbiosynthesis. In particular embodiments one or more of these genes isinactivated, such as by introduction of a mutation. In particularembodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. Inparticular embodiments, the gene encoding a transcriptional regulator offatty acid biosynthesis encodes a DNA transcription repressor, forexample, fabR.

Additionally or alternatively, said micro-organism is modified to reduceexpression of at least one of a gene encoding a pyruvate formate lyase,a gene encoding a lactate dehydrogenase, or both. In particularembodiments, the gene encoding a pyruvate formate lyase is pflB. Inparticular embodiments, the gene encoding a lactate dehydrogenase isIdhA. In particular embodiments one or more of these genes isinactivated, such as by introduction of a mutation therein.

In particular embodiments, the micro-organism is selected from the genusEscherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus,Synechoystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora,Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor,Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes,Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces,Yarrowia, or Streptomyces.

The Use of Cpf1 in the Generation of Micro-Organisms Capable of OrganicAcid Production

The methods provided herein are further used to engineer micro-organismscapable of organic acid production, more particularly from pentose orhexose sugars. In particular embodiments, the methods compriseintroducing into a micro-organism an exogenous LDH gene. In particularembodiments, the organic acid production in said micro-organisms isadditionally or alternatively increased by inactivating endogenous genesencoding proteins involved in an endogenous metabolic pathway whichproduces a metabolite other than the organic acid of interest and/orwherein the endogenous metabolic pathway consumes the organic acid. Inparticular embodiments, the modification ensures that the production ofthe metabolite other than the organic acid of interest is reduced.According to particular embodiments, the methods are used to introduceat least one engineered gene deletion and/or inactivation of anendogenous pathway in which the organic acid is consumed or a geneencoding a product involved in an endogenous pathway which produces ametabolite other than the organic acid of interest. In particularembodiments, the at least one engineered gene deletion or inactivationis in one or more gene encoding an enzyme selected from the groupconsisting of pyruvate decarboxylase (pdc), fumarate reductase, alcoholdehydrogenase (adh), acetaldehyde dehydrogenase, phosphoenolpyruvatecarboxylase (ppc), D-lactate dehydrogenase (d-ldh), L-lactatedehydrogenase (l-ldh), lactate 2-monooxygenase. In further embodimentsthe at least one engineered gene deletion and/or inactivation is in anendogenous gene encoding pyruvate decarboxylase (pdc).

In further embodiments, the micro-organism is engineered to producelactic acid and the at least one engineered gene deletion and/orinactivation is in an endogenous gene encoding lactate dehydrogenase.Additionally or alternatively, the micro-organism comprises at least oneengineered gene deletion or inactivation of an endogenous gene encodinga cytochrome-dependent lactate dehydrogenase, such as a cytochromeB2-dependent L-lactate dehydrogenase.

The Use of Cpf1 in the Generation of Improved Xylose or CellobioseUtilizing Yeasts Strains

In particular embodiments, the Cpf1 CRISPR system may be applied toselect for improved xylose or cellobiose utilizing yeast strains.Error-prone PCR can be used to amplify one (or more) genes involved inthe xylose utilization or cellobiose utilization pathways. Examples ofgenes involved in xylose utilization pathways and cellobiose utilizationpathways may include, without limitation, those described in Ha, S. J.,et al, (2011) Proc. Natl. Acad. Sci, USA 108(2):504-9 and Galazka, J.M., et al. (2010) Science 330(6000):84-6. Resulting libraries ofdouble-stranded DNA molecules, each comprising a random mutation in sucha selected gene could be co-transformed with the components of the Cpf1CRISPR system into a yeast strain (for instance S288C) and strains canbe selected with enhanced xylose or cellobiose utilization capacity, asdescribed in WO 2015138855.

The Use of Cpf1 in the Generation of Improved Yeasts Strains for Use inIsoprenoid Biosynthesis

Tadas Jakočiūnas et al. described the successful application of amultiplex CRISPR/Cas9 system for genome engineering of up to 5 differentgenomic loci in one transformation step in baker's yeast Saccharomycescerevisiae (Metabolic Engineering Volume 28, March 2015, Pages 213-222)resulting in strains with high mevalonate production, a key intermediatefor the industrially important isoprenoid biosynthesis pathway. Inparticular embodiments, the Cpf1 CRISPR system may be applied in amultiplex genome engineering method as described herein for identifyingadditional high producing yeast strains for use in isoprenoid synthesis.

The Use of Cpf1 in the Generation of Lactic Acid Producing YeastsStrains

In another embodiment, successful application of a multiplex Cpf1 CRISPRsystem is encompassed. In analogy with Vratislav Stovicek et al.(Metabolic Engineering Communications, Volume 2, December 2015, Pages13-22), improved lactic acid-producing strains can be designed andobtained in a single transformation event. In a particular embodiment,the Cpf1 CRISPR system is used for simultaneously inserting theheterologous lactate dehydrogenase gene and disruption of two endogenousgenes PDC1 and PDC5 genes.

Further Applications of the Cpf1 CRISPR System in Plants

In particular embodiments, the CRISPR system, and preferably the Cpf1CRISPR system described herein, can be used for visualization of geneticelement dynamics. For example, CRISPR imaging can visualize eitherrepetitive or non-repetitive genomic sequences, report telomere lengthchange and telomere movements and monitor the dynamics of gene locithroughout the cell cycle (Chen et al., Cell, 2013). These methods mayalso be applied to plants.

Other applications of the CRISPR system, and preferably the Cpf1 CRISPRsystem described herein, is the targeted gene disruptionpositive-selection screening in vitro and in vivo (Malina et al., Genesand Development, 2013). These methods may also be applied to plants.

In particular embodiments, fusion of inactive Cpf1 endonucleases withhistone-modifying enzymes can introduce custom changes in the complexepigenome (Rusk et al., Nature Methods, 2014). These methods may also beapplied to plants.

In particular embodiments, the CRISPR system, and preferably the Cpf1CRISPR system described herein, can be used to purify a specific portionof the chromatin and identify the associated proteins, thus elucidatingtheir regulatory roles in transcription (Waldrip et al., Epigenetics,2014). These methods may also be applied to plants.

In particular embodiments, present invention can be used as a therapyfor virus removal in plant systems as it is able to cleave both viralDNA and RNA. Previous studies in human systems have demonstrated thesuccess of utilizing CRISPR in targeting the single strand RNA virus,hepatitis C (A. Price, et al., Proc. Natl. Acad. Sci, 2015) as well asthe double stranded DNA virus, hepatitis B (V. Ramanan, et al., Sci.Rep, 2015). These methods may also be adapted for using the Cpf1 CRISPRsystem in plants.

In particular embodiments, present invention could be used to altergenome complexicity. In further particular embodiment, the CRISPRsystem, and preferably the Cpf1 CRISPR system described herein, can beused to disrupt or alter chromosome number and generate haploid plants,which only contain chromosomes from one parent. Such plants can beinduced to undergo chromosome duplication and converted into diploidplants containing only homozygous alleles (Karimi-Ashtiyani et al.,PNAS, 2015; Anton et al., Nucleus, 2014). These methods may also beapplied to plants.

In particular embodiments, the Cpf1 CRISPR system described herein, canbe used for self-cleavage. In these embodiments, the promotor of theCpf1 enzyme and gRNA can be a constitutive promotor and a second gRNA isintroduced in the same transformation cassette, but controlled by aninducible promoter. This second gRNA can be designated to inducesite-specific cleavage in the Cpf1 gene in order to create anon-functional Cpf1. In a further particular embodiment, the second gRNAinduces cleavage on both ends of the transformation cassette, resultingin the removal of the cassette from the host genome. This system offersa controlled duration of cellular exposure to the Cas enzyme and furtherminimizes off-target editing. Furthermore, cleavage of both ends of aCRISPR/Cas cassette can be used to generate transgene-free TO plantswith bi-allelic mutations (as described for Cas9 e.g. Moore et al.,Nucleic Acids Research, 2014; Schaeffer et al., Plant Science, 2015).The methods of Moore et al. may be applied to the Cpf1 CRISPR systemsdescribed herein. Sugano et al. (Plant Cell Physiol. 2014 March;55(3):475-81. doi: 10.1093/pcp/pcu014. Epub 2014 Jan. 18) reports theapplication of CRISPR-Cas9 to targeted mutagenesis in the liverwortMarchantia polymorpha L., which has emerged as a model species forstudying land plant evolution. The U6 promoter of M. polymorpha wasidentified and cloned to express the gRNA. The target sequence of thegRNA was designed to disrupt the gene encoding auxin response factor 1(ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation,Sugano et al. isolated stable mutants in the gametophyte generation ofM. polymorpha. CRISPR-Cas9-based site-directed mutagenesis in vivo wasachieved using either the Cauliflower mosaic virus 35S or M. polymorphaEF1α promoter to express Cas9. Isolated mutant individuals showing anauxin-resistant phenotype were not chimeric. Moreover, stable mutantswere produced by asexual reproduction of T1 plants. Multiple arf1alleles were easily established using CRIPSR-Cas9-based targetedmutagenesis. The methods of Sugano et al. may be applied to the Cpf1effector protein system of the present invention.

Kabadi et al. (Nucleic Acids Res. 2014 Oct. 29; 42(19):e147. doi:10.1093/nar/gku749. Epub 2014 Aug. 13) developed a single lentiviralsystem to express a Cas9 variant, a reporter gene and up to four sgRNAsfrom independent RNA polymerase III promoters that are incorporated intothe vector by a convenient Golden Gate cloning method. Each sgRNA wasefficiently expressed and can mediate multiplex gene editing andsustained transcriptional activation in immortalized and primary humancells. The methods of Kabadi et al. may be applied to the Cpf1 effectorprotein system of the present invention.

Ling et al. (BMC Plant Biology 2014, 14:327) developed a CRISPR-Cas9binary vector set based on the pGreen or pCAMBIA backbone, as well as agRNA This toolkit requires no restriction enzymes besides BsaI togenerate final constructs harboring maize-codon optimized Cas9 and oneor more gRNAs with high efficiency in as little as one cloning step. Thetoolkit was validated using maize protoplasts, transgenic maize lines,and transgenic Arabidopsis lines and was shown to exhibit highefficiency and specificity. More importantly, using this toolkit,targeted mutations of three Arabidopsis genes were detected intransgenic seedlings of the T1 generation. Moreover, the multiple-genemutations could be inherited by the next generation. (guide RNA)modulevector set, as a toolkit for multiplex genome editing in plants. Thetoolbox of Lin et al. may be applied to the Cpf1 effector protein systemof the present invention.

Protocols for targeted plant genome editing via CRISPR-Cpf1 are alsoavailable based on those disclosed for the CRISPR-Cas9 system in volume1284 of the series Methods in Molecular Biology pp 239-255 10 Feb. 2015.A detailed procedure to design, construct, and evaluate dual gRNAs forplant codon optimized Cas9 (pcoCas9) mediated genome editing usingArabidopsis thaliana and Nicotiana benthamiana protoplasts s modelcellular systems are described. Strategies to apply the CRISPR-Cas9system to generating targeted genome modifications in whole plants arealso discussed. The protocols described in the chapter may be applied tothe Cpf1 effector protein system of the present invention.

Petersen (“Towards precisely glycol engineered plants,” Plant BiotechDenmark Annual meeting 2015, Copenhagen, Denmark) developed a method ofusing CRISPR/Cas9 to engineer genome changes in Arabidopsis, for exampleto glyco engineer Arabidopsis for production of proteins and productshaving desired posttranslational modifications. Hebelstrup et al. (FrontPlant Sci. 2015 Apr. 23; 6:247) outlines in planta starchbioengineering, providing crops that express starch modifying enzymesand directly produce products that normally are made by industrialchemical and/or physical treatments of starches. The methods of Petersenand Hebelstrup may be applied to the Cpf1 effector protein system of thepresent invention.

Ma et al. (Mol Plant. 2015 Aug. 3; 8(8):1274-84. doi:10.1016/j.molp.2015.04.007) reports robust CRISPR-Cas9 vector system,utilizing a plant codon optimized Cas9 gene, for convenient andhigh-efficiency multiplex genome editing in monocot and dicot plants. Maet al. designed PCR-based procedures to rapidly generate multiple sgRNAexpression cassettes, which can be assembled into the binary CRISPR-Cas9vectors in one round of cloning by Golden Gate ligation or GibsonAssembly. With this system, Ma et al. edited 46 target sites in ricewith an average 85.4% rate of mutation, mostly in biallelic andhomozygous status. Ma et al. provide examples of loss-of-function genemutations in T0 rice and T1Arabidopsis plants by simultaneous targetingof multiple (up to eight) members of a gene family, multiple genes in abiosynthetic pathway, or multiple sites in a single gene. The methods ofMa et al. may be applied to the Cpf1 effector protein system of thepresent invention.

Lowder et al. (Plant Physiol. 2015 Aug. 21. pii: pp. 00636.2015) alsodeveloped a CRISPR-Cas9 toolbox enables multiplex genome editing andtranscriptional regulation of expressed, silenced or non-coding genes inplants. This toolbox provides researchers with a protocol and reagentsto quickly and efficiently assemble functional CRISPR-Cas9 T-DNAconstructs for monocots and dicots using Golden Gate and Gateway cloningmethods. It comes with a full suite of capabilities, includingmultiplexed gene editing and transcriptional activation or repression ofplant endogenous genes. T-DNA based transformation technology isfundamental to modern plant biotechnology, genetics, molecular biologyand physiology. As such, Applicants developed a method for the assemblyof Cas9 (WT, nickase or dCas9) and gRNA(s) into a T-DNAdestination-vector of interest. The assembly method is based on bothGolden Gate assembly and MultiSite Gateway recombination. Three modulesare required for assembly. The first module is a Cas9 entry vector,which contains promoterless Cas9 or its derivative genes flanked byattL1 and attR5 sites. The second module is a gRNA entry vector whichcontains entry gRNA expression cassettes flanked by attL5 and attL2sites. The third module includes attR1-attR2-containing destinationT-DNA vectors that provide promoters of choice for Cas9 expression. Thetoolbox of Lowder et al. may be applied to the Cpf1 effector proteinsystem of the present invention.

In an advantageous embodiment, the plant may be a tree. The presentinvention may also utilize the herein disclosed CRISPR Cas system forherbaceous systems (see, e.g., Belhaj et al., Plant Methods 9: 39 andHarrison et al., Genes & Development 28: 1859-1872). In a particularlyadvantageous embodiment, the CRISPR Cas system of the present inventionmay target single nucleotide polymorphisms (SNPs) in trees (see, e.g.,Zhou et al., New Phytologist, Volume 208, Issue 2, pages 298-301,October 2015). In the Zhou et al. study, the authors applied a CRISPRCas system in the woody perennial Populus using the 4-coumarate:CoAligase (4CL) gene family as a case study and achieved 100% mutationalefficiency for two 4CL genes targeted, with every transformant examinedcarrying biallelic modifications. In the Zhou et al., study, theCRISPR-Cas9 system was highly sensitive to single nucleotidepolymorphisms (SNPs), as cleavage for a third 4CL gene was abolished dueto SNPs in the target sequence. These methods may be applied to the Cpf1effector protein system of the present invention.

The methods of Zhou et al. (New Phytologist, Volume 208, Issue 2, pages298-301, October 2015) may be applied to the present invention asfollows. Two 4CL genes, 4CL1 and 4CL2, associated with lignin andflavonoid biosynthesis, respectively are targeted for CRISPR-Cas9editing. The Populus tremula×alba clone 717-1B4 routinely used fortransformation is divergent from the genome-sequenced Populustrichocarpa. Therefore, the 4CL1 and 4CL2 gRNAs designed from thereference genome are interrogated with in-house 717 RNA-Seq data toensure the absence of SNPs which could limit Cas efficiency. A thirdgRNA designed for 4CL5, a genome duplicate of 4CL1, is also included.The corresponding 717 sequence harbors one SNP in each allelenear/within the PAM, both of which are expected to abolish targeting bythe 4CL5-gRNA. All three gRNA target sites are located within the firstexon. For 717 transformation, the gRNA is expressed from the MedicagoU6.6 promoter, along with a human codon-optimized Cas under control ofthe CaMV 35S promoter in a binary vector. Transformation with theCas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2lines are subjected to amplicon-sequencing. The data is then processedand biallelic mutations are confirmed in all cases. These methods may beapplied to the Cpf1 effector protein system of the present invention.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f dianthii Puccinia graminis f. sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

Improved Plants and Yeast Cells

The present invention also provides plants and yeast cells obtainableand obtained by the methods provided herein. The improved plantsobtained by the methods described herein may be useful in food or feedproduction through expression of genes which, for instance ensuretolerance to plant pests, herbicides, drought, low or high temperatures,excessive water, etc.

The improved plants obtained by the methods described herein, especiallycrops and algae may be useful in food or feed production throughexpression of, for instance, higher protein, carbohydrate, nutrient orvitamin levels than would normally be seen in the wildtype. In thisregard, improved plants, especially pulses and tubers are preferred.

Improved algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

The invention also provides for improved parts of a plant. Plant partsinclude, but are not limited to, leaves, stems, roots, tubers, seeds,endosperm, ovule, and pollen. Plant parts as envisaged herein may beviable, nonviable, regeneratable, and/or non-regeneratable.

It is also encompassed herein to provide plant cells and plantsgenerated according to the methods of the invention. Gametes, seeds,embryos, either zygotic or somatic, progeny or hybrids of plantscomprising the genetic modification, which are produced by traditionalbreeding methods, are also included within the scope of the presentinvention. Such plants may contain a heterologous or foreign DNAsequence inserted at or instead of a target sequence. Alternatively,such plants may contain only an alteration (mutation, deletion,insertion, substitution) in one or more nucleotides. As such, suchplants will only be different from their progenitor plants by thepresence of the particular modification.

Thus, the invention provides a plant, animal or cell, produced by thepresent methods, or a progeny thereof. The progeny may be a clone of theproduced plant or animal, or may result from sexual reproduction bycrossing with other individuals of the same species to introgressfurther desirable traits into their offspring. The cell may be in vivoor ex vivo in the cases of multicellular organisms, particularly animalsor plants.

Cpf1 Effector Protein Complexes can be Used in Non-HumanOrganisms/Animals

In an aspect, the invention provides a non-human eukaryotic organism;preferably a multicellular eukaryotic organism, comprising a eukaryotichost cell according to any of the described embodiments. In otheraspects, the invention provides a eukaryotic organism; preferably amulticellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. The organism in someembodiments of these aspects may be an animal; for example a mammal.Also, the organism may be an arthropod such as an insect. The organismalso may be a plant. Further, the organism may be a fungus.

The present invention may also be extended to other agriculturalapplications such as, for example, farm and production animals. Forexample, pigs have many features that make them attractive as biomedicalmodels, especially in regenerative medicine. In particular, pigs withsevere combined immunodeficiency (SCID) may provide useful models forregenerative medicine, xenotransplantation (discussed also elsewhereherein), and tumor development and will aid in developing therapies forhuman SCID patients. Lee et al., (Proc Natl Acad Sci USA. 2014 May 20;111(20):7260-5) utilized a reporter-guided transcription activator-likeeffector nuclease (TALEN) system to generated targeted modifications ofrecombination activating gene (RAG) 2 in somatic cells at highefficiency, including some that affected both alleles. The Cpf1 effectorprotein may be applied to a similar system.

The methods of Lee et al., (Proc Natl Acad Sci USA. 2014 May 20;111(20):7260-5) may be applied to the present invention analogously asfollows. Mutated pigs are produced by targeted modification of RAG2 infetal fibroblast cells followed by SCNT and embryo transfer. Constructscoding for CRISPR Cas and a reporter are electroporated intofetal-derived fibroblast cells. After 48 h, transfected cells expressingthe green fluorescent protein are sorted into individual wells of a96-well plate at an estimated dilution of a single cell per well.Targeted modification of RAG2 are screened by amplifying a genomic DNAfragment flanking any CRISPR Cas cutting sites followed by sequencingthe PCR products. After screening and ensuring lack of off-sitemutations, cells carrying targeted modification of RAG2 are used forSCNT. The polar body, along with a portion of the adjacent cytoplasm ofoocyte, presumably containing the metaphase II plate, are removed, and adonor cell are placed in the perivitelline. The reconstructed embryosare then electrically porated to fuse the donor cell with the oocyte andthen chemically activated. The activated embryos are incubated inPorcine Zygote Medium 3 (PZM3) with 0.5 μM Scriptaid (S7817;Sigma-Aldrich) for 14-16 h. Embryos are then washed to remove theScriptaid and cultured in PZM3 until they were transferred into theoviducts of surrogate pigs.

The present invention is also applicable to modifying SNPs of otheranimals, such as cows. Tan et al. (Proc Natl Acad Sci USA. 2013 Oct. 8;110(41): 16526-16531) expanded the livestock gene editing toolbox toinclude transcription activator-like (TAL) effector nuclease (TALEN)-and clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid,rAAV, and oligonucleotide templates. Gene specific gRNA sequences werecloned into the Church lab gRNA vector (Addgene ID: 41824) according totheir methods (Mali P, et al. (2013) RNA-Guided Human Genome Engineeringvia Cas9. Science 339(6121):823-826). The Cas9 nuclease was providedeither by co-transfection of the hCas9 plasmid (Addgene ID: 41815) ormRNA synthesized from RCIScript-hCas9. This RCIScript-hCas9 wasconstructed by sub-cloning the XbaI-AgeI fragment from the hCas9 plasmid(encompassing the hCas9 cDNA) into the RCIScript plasmid.

Heo et al. (Stem Cells Dev. 2015 Feb. 1; 24(3):393-402. doi:10.1089/scd.2014.0278. Epub 2014 Nov. 3) reported highly efficient genetargeting in the bovine genome using bovine pluripotent cells andclustered regularly interspaced short palindromic repeat (CRISPR)/Cas9nuclease. First, Heo et al. generate induced pluripotent stem cells(iPSCs) from bovine somatic fibroblasts by the ectopic expression ofyamanaka factors and GSK3β and MEK inhibitor (2i) treatment. Heo et al.observed that these bovine iPSCs are highly similar to naïve pluripotentstem cells with regard to gene expression and developmental potential interatomas. Moreover, CRISPR-Cas9 nuclease, which was specific for thebovine NANOG locus, showed highly efficient editing of the bovine genomein bovine iPSCs and embryos.

Igenity® provides a profile analysis of animals, such as cows, toperform and transmit traits of economic traits of economic importance,such as carcass composition, carcass quality, maternal and reproductivetraits and average daily gain. The analysis of a comprehensive Igenity®profile begins with the discovery of DNA markers (most often singlenucleotide polymorphisms or SNPs). All the markers behind the Igenity®profile were discovered by independent scientists at researchinstitutions, including universities, research organizations, andgovernment entities such as USDA. Markers are then analyzed at Igenity®in validation populations. Igenity® uses multiple resource populationsthat represent various production environments and biological types,often working with industry partners from the seedstock, cow-calf,feedlot and/or packing segments of the beef industry to collectphenotypes that are not commonly available. Cattle genome databases arewidely available, see, e.g., the NAGRP Cattle Genome CoordinationProgram (http://www.animalgenome.org/cattle/maps/db.html). Thus, thepresent invention maybe applied to target bovine SNPs. One of skill inthe art may utilize the above protocols for targeting SNPs and applythem to bovine SNPs as described, for example, by Tan et al. or Heo etal.

Qingjian Zou et al. (Journal of Molecular Cell Biology Advance Accesspublished Oct. 12, 2015) demonstrated increased muscle mass in dogs bytargeting the first exon of the dog Myostatin (MSTN) gene (a negativeregulator of skeletal muscle mass). First, the efficiency of the sgRNAwas validated, using cotransfection of the sgRNA targeting MSTN with aCas9 vector into canine embryonic fibroblasts (CEFs). Thereafter, MSTNKO dogs were generated by micro-injecting embryos with normal morphologywith a mixture of Cas9 mRNA and MSTN sgRNA and auto-transplantation ofthe zygotes into the oviduct of the same female dog. The knock-outpuppies displayed an obvious muscular phenotype on thighs compared withits wild-type littermate sister. This can also be performed using theCpf1 CRISPR systems provided herein.

Livestock—Pigs

Viral targets in livestock may include, in some embodiments, porcineCD163, for example on porcine macrophages. CD163 is associated withinfection (thought to be through viral cell entry) by PRRSv (PorcineReproductive and Respiratory Syndrome virus, an arterivirus). Infectionby PRRSv, especially of porcine alveolar macrophages (found in thelung), results in a previously incurable porcine syndrome (“Mysteryswine disease” or “blue ear disease”) that causes suffering, includingreproductive failure, weight loss and high mortality rates in domesticpigs. Opportunistic infections, such as enzootic pneumonia, meningitisand ear oedema, are often seen due to immune deficiency through loss ofmacrophage activity. It also has significant economic and environmentalrepercussions due to increased antibiotic use and financial loss (anestimated $660m per year).

As reported by Kristin M Whitworth and Dr Randall Prather et al. (NatureBiotech 3434 published online 7 Dec. 2015) at the University of Missouriand in collaboration with Genus Plc, CD163 was targeted usingCRISPR-Cas9 and the offspring of edited pigs were resistant when exposedto PRRSv. One founder male and one founder female, both of whom hadmutations in exon 7 of CD163, were bred to produce offspring. Thefounder male possessed an 11-bp deletion in exon 7 on one allele, whichresults in a frameshift mutation and missense translation at amino acid45 in domain 5 and a subsequent premature stop codon at amino acid 64.The other allele had a 2-bp addition in exon 7 and a 377-bp deletion inthe preceding intron, which were predicted to result in the expressionof the first 49 amino acids of domain 5, followed by a premature stopcode at amino acid 85. The sow had a 7 bp addition in one allele thatwhen translated was predicted to express the first 48 amino acids ofdomain 5, followed by a premature stop codon at amino acid 70. The sow'sother allele was unamplifiable. Selected offspring were predicted to bea null animal (CD163−/−), i.e. a CD163 knock out.

Accordingly, in some embodiments, porcine alveolar macrophages may betargeted by the CRISPR protein. In some embodiments, porcine CD163 maybe targeted by the CRISPR protein. In some embodiments, porcine CD163may be knocked out through induction of a DSB or through insertions ordeletions, for example targeting deletion or modification of exon 7,including one or more of those described above, or in other regions ofthe gene, for example deletion or modification of exon 5.

An edited pig and its progeny are also envisaged, for example a CD163knock out pig. This may be for livestock, breeding or modelling purposes(i.e. a porcine model). Semen comprising the gene knock out is alsoprovided.

CD163 is a member of the scavenger receptor cysteine-rich (SRCR)superfamily. Based on in vitro studies SRCR domain 5 of the protein isthe domain responsible for unpackaging and release of the viral genome.As such, other members of the SRCR superfamily may also be targeted inorder to assess resistance to other viruses. PRRSV is also a member ofthe mammalian arterivirus group, which also includes murine lactatedehydrogenase-elevating virus, simian hemorrhagic fever virus and equinearteritis virus. The arteriviruses share important pathogenesisproperties, including macrophage tropism and the capacity to cause bothsevere disease and persistent infection. Accordingly, arteriviruses, andin particular murine lactate dehydrogenase-elevating virus, simianhemorrhagic fever virus and equine arteritis virus, may be targeted, forexample through porcine CD163 or homologues thereof in other species,and murine, simian and equine models and knockout also provided.

Indeed, this approach may be extended to viruses or bacteria that causeother livestock diseases that may be transmitted to humans, such asSwine Influenza Virus (SIV) strains which include influenza C and thesubtypes of influenza A known as H1N1, H1N2, H2N1, H3N1, H3N2, and H2N3,as well as pneumonia, meningitis and oedema mentioned above.

Therapeutic Targeting with RNA-Guided Cpf1 Effector Protein Complex

As will be apparent, it is envisaged that the present system can be usedto target any polynucleotide sequence of interest. The inventionprovides a non-naturally occurring or engineered composition, or one ormore polynucleotides encoding components of said composition, or vectoror delivery systems comprising one or more polynucleotides encodingcomponents of said composition for use in a modifying a target cell invivo, ex vivo or in vitro and, may be conducted in a manner alters thecell such that once modified the progeny or cell line of the CRISPRmodified cell retains the altered phenotype. The modified cells andprogeny may be part of a multi-cellular organism such as a plant oranimal with ex vivo or in vivo application of CRISPR system to desiredcell types. The CRISPR invention may be a therapeutic method oftreatment. The therapeutic method of treatment may comprise gene orgenome editing, or gene therapy.

Treating Pathogens, Like Bacterial, Fungal and Parasitic Pathogens

The present invention may also be applied to treat bacterial, fungal andparasitic pathogens. Most research efforts have focused on developingnew antibiotics, which once developed, would nevertheless be subject tothe same problems of drug resistance. The invention provides novelCRISPR-based alternatives which overcome those difficulties.Furthermore, unlike existing antibiotics, CRISPR-based treatments can bemade pathogen specific, inducing bacterial cell death of a targetpathogen while avoiding beneficial bacteria.

Jiang et al. (“RNA-guided editing of bacterial genomes using CRISPR-Cassystems,” Nature Biotechnology vol. 31, p. 233-9, March 2013) used aCRISPR-Cas9 system to mutate or kill S. pneumoniae and E. coli. Thework, which introduced precise mutations into the genomes, relied ondual-RNA:Cas9-directed cleavage at the targeted genomic site to killunmutated cells and circumvented the need for selectable markers orcounter-selection systems. CRISPR systems have be used to reverseantibiotic resistance and eliminate the transfer of resistance betweenstrains. Bickard et al. showed that Cas9, reprogrammed to targetvirulence genes, kills virulent, but not avirulent, S. aureus.Reprogramming the nuclease to target antibiotic resistance genesdestroyed staphylococcal plasmids that harbor antibiotic resistancegenesand immunized against the spread of plasmid-borne resistance genes.(see, Bikard et al., “Exploiting CRISPR-Cas nucleases to producesequence-specific antimicrobials,” Nature Biotechnology vol. 32,1146-1150, doi:10.1038/nbt.3043, published online 5 Oct. 2014.) Bikardshowed that CRISPR-Cas9 antimicrobials function in vivo to kill S.aureus in a mouse skin colonization model. Similarly, Yosef et al used aCRISPR system to target genes encoding enzymes that confer resistance toβ-lactam antibiotics (see Yousef et al., “Temperate and lyticbacteriophages programmed to sensitize and kill antibiotic-resistantbacteria,” Proc. Natl. Acad. Sci. USA, vol. 112, p. 7267-7272, doi:10.1073/pnas.1500107112 published online May 18, 2015).

CRISPR systems can be used to edit genomes of parasites that areresistant to other genetic approaches. For example, a CRISPR-Cas9 systemwas shown to introduce double-stranded breaks into the in the Plasmodiumyoelii genome (see, Zhang et al., “Efficient Editing of Malaria ParasiteGenome Using the CRISPR/Cas9 System,” mBio. vol. 5, e01414-14,July-August 2014). Ghorbal et al (“Genome editing in the human malariaparasite Plasmodium falciparumusing the CRISPR-Cas9 system,” NatureBiotechnology, vol. 32, p. 819-821, doi: 10.1038/nbt.2925, publishedonline Jun. 1, 2014) modified the sequences of two genes, orc1 andkelch13, which have putative roles in gene silencing and emergingresistance to artemisinin, respectively. Parasites that were altered atthe appropriate sites were recovered with very high efficiency, despitethere being no direct selection for the modification, indicating thatneutral or even deleterious mutations can be generated using thissystem. CRISPR-Cas9 is also used to modify the genomes of otherpathogenic parasites, including Toxoplasma gondii (see Shen et al.,“Efficient gene disruption in diverse strains of Toxoplasma gondii usingCRISPR/CAS9,” mBio vol. 5:e01114-14, 2014; and Sidik et al., “EfficientGenome Engineering of Toxoplasma gondii Using CRISPR/Cas9,” PLoS Onevol. 9, e100450, doi: 10.1371/journal.pone.0100450, published onlineJun. 27, 2014).

Vyas et al. (“A Candida albicans CRISPR system permits geneticengineering of essential genes and gene families,” Science Advances,vol. 1, e1500248, DOI: 10.1126/sciadv.1500248, Apr. 3, 2015) employed aCRISPR system to overcome long-standing obstacles to genetic engineeringin C. albicans and efficiently mutate in a single experiment both copiesof several different genes. In an organism where several mechanismscontribute to drug resistance, Vyas produced homozygous double mutantsthat no longer displayed the hyper-resistance to fluconazole orcycloheximide displayed by the parental clinical isolate Can90. Vyasalso obtained homozygous loss-of-function mutations in essential genesof C. albicans by creating conditional alleles. Null alleles of DCR1,which is required for ribosomal RNA processing, are lethal at lowtemperature but viable at high temperature. Vyas used a repair templatethat introduced a nonsense mutation and isolated dcr1/dcr1 mutants thatfailed to grow at 16° C.

The CRISPR system of the present invention for use in P. falciparum bydisrupting chromosomal loci. Ghorbal et al. (“Genome editing in thehuman malaria parasite Plasmodium falciparum using the CRISPR-Cas9system”, Nature Biotechnology, 32, 819-821 (2014), DOI:10.1038/nbt.2925, Jun. 1, 2014) employed a CRISPR system to introducespecific gene knockouts and single-nucleotide substitutions in themalaria genome. To adapt the CRISPR-Cas9 system to P. falciparum,Ghorbal et al. generated expression vectors for under the control ofplasmodial regulatory elements in the puF1-Cas9 episome that alsocarries the drug-selectable marker ydhodh, which gives resistance toDSM-1, a P. falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitorand for transcription of the sgRNA, used P. falciparum U6 small nuclear(sn)RNA regulatory elements placing the guide RNA and the donor DNAtemplate for homologous recombination repair on the same plasmid, pL7.See also, Zhang C. et al. (“Efficient editing of malaria parasite genomeusing the CRISPR/Cas9 system”, MBio, 2014 Jul. 1; (4): E01414-14, doi:10.1128/MbIO.01414-14) and Wagner et al. (“EfficientCRISPR-Cas9-mediated genome editing in Plasmodium falciparum, NatureMethods 11, 915-918 (2014), DOI: 10.1038/nmeth.3063).

Treating Pathogens, Like Viral Pathogens Such as HIV

Cas-mediated genome editing might be used to introduce protectivemutations in somatic tissues to combat nongenetic or complex diseases.For example, NHEJ-mediated inactivation of the CCR5 receptor inlymphocytes (Lombardo et al., Nat Biotechnol. 2007 November;25(11):1298-306) may be a viable strategy for circumventing HIVinfection, whereas deletion of PCSK9 (Cohen et al., Nat Genet. 2005February; 37(2):161-5) orangiopoietin (Musunuru et al., N Engl J Med.2010 Dec. 2; 363(23):2220-7) may provide therapeutic effects againststatin-resistant hypercholesterolemia or hyperlipidemia. Although thesetargets may be also addressed using siRNA-mediated protein knockdown, aunique advantage of NHEJ-mediated gene inactivation is the ability toachieve permanent therapeutic benefit without the need for continuingtreatment. As with all gene therapies, it will of course be important toestablish that each proposed therapeutic use has a favorablebenefit-risk ratio.

Hydrodynamic delivery of plasmid DNA encoding Cas9 nd guide RNA alongwith a repair template into the liver of an adult mouse model oftyrosinemia was shown to be able to correct the mutant Fah gene andrescue expression of the wild-type Fah protein in ˜1 out of 250 cells(Nat Biotechnol. 2014 June; 32(6):551-3). In addition, clinical trialssuccessfully used ZF nucleases to combat HIV infection by ex vivoknockout of the CCR5 receptor. In all patients, HIV DNA levelsdecreased, and in one out of four patients, HIV RNA became undetectable(Tebas et al., N Engl J Med. 2014 Mar. 6; 370(10):901-10). Both of theseresults demonstrate the promise of programmable nucleases as a newtherapeutic platform.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the CRISPR-Cas system of the presentinvention. A minimum of 2.5×10⁶ CD34+ cells per kilogram patient weightmay be collected and prestimulated for 16 to 20 hours in X-VIVO 15medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

With the knowledge in the art and the teachings in this disclosure theskilled person can correct HSCs as to immunodeficiency condition such asHIV/AIDS comprising contacting an HSC with a CRISPR-Cas9 system thattargets and knocks out CCR5. An guide RNA (and advantageously a dualguide approach, e.g., a pair of different guide RNAs; for instance,guide RNAs targeting of two clinically relevant genes, B2M and CCR5, inprimary human CD4+ T cells and CD34+ hematopoietic stem and progenitorcells (HSPCs)) that targets and knocks out CCR5-and-Cpf1 proteincontaining particle is contacted with HSCs. The so contacted cells canbe administered; and optionally treated/expanded; cf. Cartier. See alsoKiem, “Hematopoietic stem cell-based gene therapy for HIV disease,” CellStem Cell. Feb. 3, 2012; 10(2): 137-147; incorporated herein byreference along with the documents it cites; Mandal et al, “EfficientAblation of Genes in Human Hematopoietic Stem and Effector Cells usingCRISPR/Cas9,” Cell Stem Cell, Volume 15, Issue 5, p643-652, 6 Nov. 2014;incorporated herein by reference along with the documents it cites.Mention is also made of Ebina, “CRISPR/Cas9 system to suppress HIV-1expression by editing HIV-1 integrated proviral DNA” SCIENTIFIC REPORTS|3: 2510|DOI: 10.1038/srep02510, incorporated herein by reference alongwith the documents it cites, as another means for combatting HIV/AIDSusing a CRISPR-Cpf1 system.

The rationale for genome editing for HIV treatment originates from theobservation that individuals homozygous for loss of function mutationsin CCR5, a cellular co-receptor for the virus, are highly resistant toinfection and otherwise healthy, suggesting that mimicking this mutationwith genome editing could be a safe and effective therapeutic strategy[Liu, R., et al. Cell 86, 367-377 (1996)]. This idea was clinicallyvalidated when an HIV infected patient was given an allogeneic bonemarrow transplant from a donor homozygous for a loss of function CCR5mutation, resulting in undetectable levels of HIV and restoration ofnormal CD4 T-cell counts [Hutter, G., et al. The New England journal ofmedicine 360, 692-698 (2009)]. Although bone marrow transplantation isnot a realistic treatment strategy for most HIV patients, due to costand potential graft vs. host disease, HIV therapies that convert apatient's own T-cells into CCR5 are desirable.

Early studies using ZFNs and NHEJ to knockout CCR5 in humanized mousemodels of HIV showed that transplantation of CCR5 edited CD4 T cellsimproved viral load and CD4 T-cell counts [Perez, E. E., et al. Naturebiotechnology 26, 808-816 (2008)]. Importantly, these models also showedthat HIV infection resulted in selection for CCR5 null cells, suggestingthat editing confers a fitness advantage and potentially allowing asmall number of edited cells to create a therapeutic effect.

As a result of this and other promising preclinical studies, genomeediting therapy that knocks out CCR5 in patient T cells has now beentested in humans [Holt, N., et al. Nature biotechnology 28, 839-847(2010); Li, L., et al. Molecular therapy: the journal of the AmericanSociety of Gene Therapy 21, 1259-1269 (2013)]. In a recent phase Iclinical trial, CD4+ T cells from patients with HIV were removed, editedwith ZFNs designed to knockout the CCR5 gene, and autologouslytransplanted back into patients [Tebas, P., et al. The New Englandjournal of medicine 370, 901-910 (2014)].

In another study (Mandal et al., Cell Stem Cell, Volume 15, Issue 5,p643-652, 6 Nov. 2014), CRISPR-Cas9 has targeted two clinical relevantgenes, B2M and CCR5, in human CD4+ T cells and CD34+ hematopoietic stemand progenitor cells (HSPCs). Use of single RNA guides led to highlyefficient mutagenesis in HSPCs but not in T cells. A dual guide approachimproved gene deletion efficacy in both cell types. HSPCs that hadundergone genome editing with CRISPR-Cas9 retained multilineagepotential. Predicted on- and off-target mutations were examined viatarget capture sequencing in HSPCs and low levels of off-targetmutagenesis were observed at only one site. These results demonstratethat CRISPR-Cas9 can efficiently ablate genes in HSPCs with minimaloff-target mutagenesis, which have broad applicability for hematopoieticcell-based therapy.

Wang et al. (PLoS One. 2014 Dec. 26; 9(12):e115987. doi:10.1371/journal.pone.0115987) silenced CCR5 via CRISPR associatedprotein 9 (Cas9) and single guided RNAs (guide RNAs) with lentiviralvectors expressing Cas9 and CCR5 guide RNAs. Wang et al. showed that asingle round transduction of lentiviral vectors expressing Cas9 and CCR5guide RNAs into HIV-1 susceptible human CD4+ cells yields highfrequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are notonly resistant to R5-tropic HIV-1, including transmitted/founder (T/F)HIV-1 isolates, but also have selective advantage over CCR5gene-undisrupted cells during R5-tropic HIV-1 infection. Genomemutations at potential off-target sites that are highly homologous tothese CCR5 guide RNAs in stably transduced cells even at 84 days posttransduction were not detected by a T7 endonuclease I assay.

Fine et al. (Sci Rep. 2015 Jul. 1; 5:10777. doi: 10.1038/srep10777)identified a two-cassette system expressing pieces of the S. pyogenesCas9 (SpCas9) protein which splice together in cellula to form afunctional protein capable of site-specific DNA cleavage. With specificCRISPR guide strands, Fine et al. demonstrated the efficacy of thissystem in cleaving the HBB and CCR5 genes in human HEK-293T cells as asingle Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9(tsSpCas9) displayed ˜35% of the nuclease activity compared with thewild-type SpCas9 (wtSpCas9) at standard transfection doses, but hadsubstantially decreased activity at lower dosing levels. The greatlyreduced open reading frame length of the tsSpCas9 relative to wtSpCas9potentially allows for more complex and longer genetic elements to bepackaged into an AAV vector including tissue-specific promoters,multiplexed guide RNA expression, and effector domain fusions to SpCas9.

Li et al. (J Gen Virol. 2015 August; 96(8):2381-93. doi:10.1099/vir.0.000139. Epub 2015 Apr. 8) demonstrated that CRISPR-Cas9can efficiently mediate the editing of the CCR5 locus in cell lines,resulting in the knockout of CCR5 expression on the cell surface.Next-generation sequencing revealed that various mutations wereintroduced around the predicted cleavage site of CCR5. For each of thethree most effective guide RNAs that were analyzed, no significantoff-target effects were detected at the 15 top-scoring potential sites.By constructing chimeric Ad5F35 adenoviruses carrying CRISPR-Cas9components, Li et al. efficiently transduced primary CD4+ T-lymphocytesand disrupted CCR5 expression, and the positively transduced cells wereconferred with HIV-1 resistance.

One of skill in the art may utilize the above studies of, for example,Holt, N., et al. Nature biotechnology 28, 839-847 (2010), Li, L., et al.Molecular therapy: the journal of the American Society of Gene Therapy21, 1259-1269 (2013), Mandal et al., Cell Stem Cell, Volume 15, Issue 5,p643-652, 6 Nov. 2014, Wang et al. (PLoS One. 2014 Dec. 26;9(12):e115987. doi: 10.1371/journal.pone.0115987), Fine et al. (Sci Rep.2015 Jul. 1; 5:10777. doi: 10.1038/srep10777) and Li et al. (J GenVirol. 2015 August; 96(8):2381-93. doi: 10.1099/vir.0.000139. Epub 2015Apr. 8) for targeting CCR5 with the CRISPR Cas system of the presentinvention.

Treating Pathogens, Like Viral Pathogens, Such as HBV

The present invention may also be applied to treat hepatitis B virus(HBV). However, the CRISPR Cas system must be adapted to avoid theshortcomings of RNAi, such as the risk of oversatring endogenous smallRNA pathways, by for example, optimizing dose and sequence (see, e.g.,Grimm et al., Nature vol. 441, 26 May 2006). For example, low doses,such as about 1-10×10¹⁴ particles per human are contemplated. In anotherembodiment, the CRISPR Cas system directed against HBV may beadministered in liposomes, such as a stable nucleic-acid-lipid particle(SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No.8, August 2005). Daily intravenous injections of about 1, 3 or 5mg/kg/day of CRISPR Cas targeted to HBV RNA in a SNALP are contemplated.The daily treatment may be over about three days and then weekly forabout five weeks. In another embodiment, the system of Chen et al. (GeneTherapy (2007) 14, 11-19) may be used/and or adapted for the CRISPR Cassystem of the present invention. Chen et al. use a double-strandedadenoassociated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA.A single administration of dsAAV2/8 vector (1×10¹² vector genomes permouse), carrying HBV-specific shRNA, effectively suppressed the steadylevel of HBV protein, mRNA and replicative DNA in liver of HBVtransgenic mice, leading to up to 2-3 log₁₀ decrease in HBV load in thecirculation. Significant HBV suppression sustained for at least 120 daysafter vector administration. The therapeutic effect of shRNA was targetsequence dependent and did not involve activation of interferon. For thepresent invention, a CRISPR Cas system directed to HBV may be clonedinto an AAV vector, such as a dsAAV2/8 vector and administered to ahuman, for example, at a dosage of about 1×10¹⁵ vector genomes to about1×10¹⁶ vector genomes per human. In another embodiment, the method ofWooddell et al. (Molecular Therapy vol. 21 no. 5, 973-985 May 2013) maybe used/and or adapted to the CRISPR Cas system of the presentinvention. Woodell et al. show that simple coinjection of ahepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-likepeptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA(chol-siRNA) targeting coagulation factor VII (F7) results in efficientF7 knockdown in mice and nonhuman primates without changes in clinicalchemistry or induction of cytokines. Using transient and transgenicmouse models of HBV infection, Wooddell et al. show that a singlecoinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBVsequences resulted in multilog repression of viral RNA, proteins, andviral DNA with long duration of effect. Intraveinous coinjections, forexample, of about 6 mg/kg of NAG-MLP and 6 mg/kg of HBV specific CRISPRCas may be envisioned for the present invention. In the alternative,about 3 mg/kg of NAG-MLP and 3 mg/kg of HBV specific CRISPR Cas may bedelivered on day one, followed by administration of about 2-3 mg/kg ofNAG-MLP and 2-3 mg/kg of HBV specific CRISPR Cas two weeks later.

Lin et al. (Mol Ther Nucleic Acids. 2014 Aug. 19; 3:e186. doi:10.1038/mtna.2014.38) designed eight gRNAs against HBV of genotype A.With the HBV-specific gRNAs, the CRISPR-Cas9 system significantlyreduced the production of HBV core and surface proteins in Huh-7 cellstransfected with an HBV-expression vector. Among eight screened gRNAs,two effective ones were identified. One gRNA targeting the conserved HBVsequence acted against different genotypes. Using a hydrodynamics-HBVpersistence mouse model, Lin et al. further demonstrated that thissystem could cleave the intrahepatic HBV genome-containing plasmid andfacilitate its clearance in vivo, resulting in reduction of serumsurface antigen levels. These data suggest that the CRISPR-Cas9 systemcould disrupt the HBV-expressing templates both in vitro and in vivo,indicating its potential in eradicating persistent HBV infection.

Dong et al. (Antiviral Res. 2015 June; 118:110-7. doi:10.1016/j.antiviral.2015.03.015. Epub 2015 Apr. 3) used the CRISPR-Cas9system to target the HBV genome and efficiently inhibit HBV infection.Dong et al. synthesized four single-guide RNAs (guide RNAs) targetingthe conserved regions of HBV. The expression of these guide RNAS withCas9 reduced the viral production in Huh7 cells as well as inHBV-replication cell HepG2.2.15. Dong et al. further demonstrated thatCRISPR-Cas9 direct cleavage and cleavage-mediated mutagenesis occurredin HBV cccDNA of transfected cells. In the mouse model carrying HBVcccDNA, injection of guide RNA-Cas9 plasmids via rapid tail veinresulted in the low level of cccDNA and HBV protein.

Liu et al. (J Gen Virol. 2015 August; 96(8):2252-61. doi:10.1099/vir.0.000159. Epub 2015 Apr. 22) designed eight guide RNAs(gRNAs) that targeted the conserved regions of different HBV genotypes,which could significantly inhibit HBV replication both in vitro and invivo to investigate the possibility of using the CRISPR-Cas9 system todisrupt the HBV DNA templates. The HBV-specific gRNA/Cpf1 system couldinhibit the replication of HBV of different genotypes in cells, and theviral DNA was significantly reduced by a single gRNA/Cpf1 system andcleared by a combination of different gRNA/Cpf1 systems.

Wang et al. (World J Gastroenterol. 2015 Aug. 28; 21(32):9554-65. doi:10.3748/wjg.v21.i32.9554) designed 15 gRNAs against HBV of genotypesA-D. Eleven combinations of two above gRNAs (dual-gRNAs) covering theregulatory region of HBV were chosen. The efficiency of each gRNA and 11dual-gRNAs on the suppression of HBV (genotypes A-D) replication wasexamined by the measurement of HBV surface antigen (HBsAg) or e antigen(HBeAg) in the culture supernatant. The destruction of HBV-expressingvector was examined in HuH7 cells co-transfected with dual-gRNAs andHBV-expressing vector using polymerase chain reaction (PCR) andsequencing method, and the destruction of cccDNA was examined in HepAD38cells using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD)digestion, rolling circle amplification and quantitative PCR combinedmethod. The cytotoxicity of these gRNAs was assessed by a mitochondrialtetrazolium assay. All of gRNAs could significantly reduce HBsAg orHBeAg production in the culture supernatant, which was dependent on theregion in which gRNA against. All of dual gRNAs could efficientlysuppress HBsAg and/or HBeAg production for HBV of genotypes A-D, and theefficacy of dual gRNAs in suppressing HBsAg and/or HBeAg production wassignificantly increased when compared to the single gRNA used alone.Furthermore, by PCR direct sequencing we confirmed that these dual gRNAscould specifically destroy HBV expressing template by removing thefragment between the cleavage sites of the two used gRNAs. Mostimportantly, gRNA-5 and gRNA-12 combination not only could efficientlysuppressing HBsAg and/or HBeAg production, but also destroy the cccDNAreservoirs in HepAD38 cells.

Karimova et al. (Sci Rep. 2015 Sep. 3; 5:13734. doi: 10.1038/srep13734)identified cross-genotype conserved HBV sequences in the S and X regionof the HBV genome that were targeted for specific and effective cleavageby a Cas9 nickase. This approach disrupted not only episomal cccDNA andchromosomally integrated HBV target sites in reporter cell lines, butalso HBV replication in chronically and de novo infected hepatoma celllines.

One of skill in the art may utilize the above studies of, for example,Lin et al. (Mol Ther Nucleic Acids. 2014 Aug. 19; 3:e186. doi:10.1038/mtna.2014.38), Dong et al. (Antiviral Res. 2015 June; 118:110-7.doi: 10.1016/j.antiviral.2015.03.015. Epub 2015 Apr. 3), Liu et al. (JGen Virol. 2015 August; 96(8):2252-61. doi: 10.1099/vir.0.000159. Epub2015 Apr. 22), Wang et al. (World J Gastroenterol. 2015 Aug. 28;21(32):9554-65. doi: 10.3748/wjg.v21.i32.9554) and Karimova et al. (SciRep. 2015 Sep. 3; 5:13734. doi: 10.1038/srep13734) for targeting HBVwith the CRISPR Cas system of the present invention.

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, andseldom cured due to the persistence of viral episomal DNA (cccDNA) ininfected cells. Ramanan et al. (Ramanan V, Shlomai A, Cox D B, SchwartzR E, Michailidis E, Bhatta A, Scott D A, Zhang F, Rice C M, Bhatia S N,Sci Rep. 2015 Jun. 2; 5:10833. doi: 10.1038/srep10833, published online2 Jun. 2015) showed that the CRISPR/Cas9 system can specifically targetand cleave conserved regions in the HBV genome, resulting in robustsuppression of viral gene expression and replication. Upon sustainedexpression of Cas9 and appropriately chosen guide RNAs, theydemonstrated cleavage of cccDNA by Cas9 and a dramatic reduction in bothcccDNA and other parameters of viral gene expression and replication.Thus, they showed that directly targeting viral episomal DNA is a noveltherapeutic approach to control the virus and possibly cure patients.This is also described in WO2015089465 A1, in the name of The BroadInstitute et al., the contents of which are hereby incorporated byreference

As such targeting viral episomal DNA in HBV is preferred in someembodiments.

The present invention may also be applied to treat pathogens, e.g.bacterial, fungal and parasitic pathogens. Most research efforts havefocused on developing new antibiotics, which once developed, wouldnevertheless be subject to the same problems of drug resistance. Theinvention provides novel CRISPR-based alternatives which overcome thosedifficulties. Furthermore, unlike existing antibiotics, CRISPR-basedtreatments can be made pathogen specific, inducing bacterial cell deathof a target pathogen while avoiding beneficial bacteria.

The present invention may also be applied to treat hepatitis C virus(HCV). The methods of Roelvinki et al. (Molecular Therapy vol. 20 no. 9,1737-1749 September 2012) may be applied to the CRISPR Cas system. Forexample, an AAV vector such as AAV8 may be a contemplated vector and forexample a dosage of about 1.25×1011 to 1.25×1013 vector genomes perkilogram body weight (vg/kg) may be contemplated. The present inventionmay also be applied to treat pathogens, e.g. bacterial, fungal andparasitic pathogens. Most research efforts have focused on developingnew antibiotics, which once developed, would nevertheless be subject tothe same problems of drug resistance. The invention provides novelCRISPR-based alternatives which overcome those difficulties.Furthermore, unlike existing antibiotics, CRISPR-based treatments can bemade pathogen specific, inducing bacterial cell death of a targetpathogen while avoiding beneficial bacteria.

Jiang et al. (“RNA-guided editing of bacterial genomes using CRISPR-Cassystems,” Nature Biotechnology vol. 31, p. 233-9, March 2013) used aCRISPR-Cas9 system to mutate or kill S. pneumoniae and E. coli. Thework, which introduced precise mutations into the genomes, relied ondual-RNA:Cas9-directed cleavage at the targeted genomic site to killunmutated cells and circumvented the need for selectable markers orcounter-selection systems. CRISPR systems have be used to reverseantibiotic resistance and eliminate the transfer of resistance betweenstrains. Bickard et al. showed that Cas9, reprogrammed to targetvirulence genes, kills virulent, but not avirulent, S. aureus.Reprogramming the nuclease to target antibiotic resistance genesdestroyed staphylococcal plasmids that harbor antibiotic resistancegenesand immunized against the spread of plasmid-borne resistance genes.(see, Bikard et al., “Exploiting CRISPR-Cas nucleases to producesequence-specific antimicrobials,” Nature Biotechnology vol. 32,1146-1150, doi:10.1038/nbt.3043, published online 5 Oct. 2014.) Bikardshowed that CRISPR-Cas9 antimicrobials function in vivo to kill S.aureus in a mouse skin colonization model. Similarly, Yosef et al used aCRISPR system to target genes encoding enzymes that confer resistance toβ-lactam antibiotics (see Yousef et al., “Temperate and lyticbacteriophages programmed to sensitize and kill antibiotic-resistantbacteria,” Proc. Natl. Acad. Sci. USA, vol. 112, p. 7267-7272, doi:10.1073/pnas.1500107112 published online May 18, 2015).

CRISPR systems can be used to edit genomes of parasites that areresistant to other genetic approaches. For example, a CRISPR-Cas9 systemwas shown to introduce double-stranded breaks into the in the Plasmodiumyoelii genome (see, Zhang et al., “Efficient Editing of Malaria ParasiteGenome Using the CRISPR/Cas9 System,” mBio. vol. 5, e01414-14,July-August 2014). Ghorbal et al. (“Genome editing in the human malariaparasite Plasmodium falciparumusing the CRISPR-Cas9 system,” NatureBiotechnology, vol. 32, p. 819-821, doi: 10.1038/nbt.2925, publishedonline Jun. 1, 2014) modified the sequences of two genes, orc1 andkelch13, which have putative roles in gene silencing and emergingresistance to artemisinin, respectively. Parasites that were altered atthe appropriate sites were recovered with very high efficiency, despitethere being no direct selection for the modification, indicating thatneutral or even deleterious mutations can be generated using thissystem. CRISPR-Cas9 is also used to modify the genomes of otherpathogenic parasites, including Toxoplasma gondii (see Shen et al.,“Efficient gene disruption in diverse strains of Toxoplasma gondii usingCRISPR/CAS9,” mBio vol. 5:e01114-14, 2014; and Sidik et al., “EfficientGenome Engineering of Toxoplasma gondii Using CRISPR/Cas9,” PLoS Onevol. 9, e100450, doi: 10.1371/journal.pone.0100450, published onlineJun. 27, 2014).

Vyas et al. (“A Candida albicans CRISPR system permits geneticengineering of essential genes and gene families,” Science Advances,vol. 1, e1500248, DOI: 10.1126/sciadv.1500248, Apr. 3, 2015) employed aCRISPR system to overcome long-standing obstacles to genetic engineeringin C. albicans and efficiently mutate in a single experiment both copiesof several different genes. In an organism where several mechanismscontribute to drug resistance, Vyas produced homozygous double mutantsthat no longer displayed the hyper-resistance to fluconazole orcycloheximide displayed by the parental clinical isolate Can90. Vyasalso obtained homozygous loss-of-function mutations in essential genesof C. albicans by creating conditional alleles. Null alleles of DCR1,which is required for ribosomal RNA processing, are lethal at lowtemperature but viable at high temperature. Vyas used a repair templatethat introduced a nonsense mutation and isolated dcr1/dcr1 mutants thatfailed to grow at 16° C.

Treating Diseases with Genetic or Epigenetic Aspects

The CRISPR-Cas systems of the present invention can be used to correctgenetic mutations that were previously attempted with limited successusing TALEN and ZFN and have been identified as potential targets forCas9 systems, including as in published applications of Editas Medicinedescribing methods to use Cas9 systems to target loci to therapeuticallyaddress diseases with gene therapy, including, WO 2015/048577CRISPR-RELATED METHODS AND COMPOSITIONS of Gluckmann et al.; WO2015/070083 CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNASof Glucksmann et al.; In some embodiments, the treatment, prophylaxis ordiagnosis of Primary Open Angle Glaucoma (POAG) is provided. The targetis preferably the MYOC gene. This is described in WO2015153780, thedisclosure of which is hereby incorporated by reference.

Mention is made of WO2015/134812 CRISPR/CAS-RELATED METHODS ANDCOMPOSITIONS FOR TREATING USHER SYNDROME AND RETINITIS PIGMENTOSA ofMaeder et al. Through the teachings herein the invention comprehendsmethods and materials of these documents applied in conjunction with theteachings herein. In an aspect of ocular and auditory gene therapy,methods and compositions for treating Usher Syndrome andRetinis-Pigmentosa may be adapted to the CRISPR-Cas system of thepresent invention (see, e.g., WO 2015/134812). In an embodiment, the WO2015/134812 involves a treatment or delaying the onset or progression ofUsher Syndrome type IIA (USH2A, USH11A) and retinitis pigmentosa 39(RP39) by gene editing, e.g., using CRISPR-Cas9 mediated methods tocorrect the guanine deletion at position 2299 in the USH2A gene (e.g.,replace the deleted guanine residue at position 2299 in the USH2A gene).A similar effect can be achieved with Cpf1. In a related aspect, amutation is targeted by cleaving with either one or more nuclease, oneor more nickase, or a combination thereof, e.g., to induce HDR with adonor template that corrects the point mutation (e.g., the singlenucleotide, e.g., guanine, deletion). The alteration or correction ofthe mutant USH2A gene can be mediated by any mechanism. Exemplarymechanisms that can be associated with the alteration (e.g., correction)of the mutant HSH2A gene include, but are not limited to, non-homologousend joining, microhomology-mediated end joining (MMEJ),homology-directed repair (e.g., endogenous donor template mediated),SDSA (synthesis dependent strand annealing), single-strand annealing orsingle strand invasion. In an embodiment, the method used for treatingUsher Syndrome and Retinis-Pigmentosa can include acquiring knowledge ofthe mutation carried by the subject, e.g., by sequencing the appropriateportion of the USH2A gene.

Mention is also made of WO 2015/138510 and through the teachings hereinthe invention (using a CRISPR-Cas9 system) comprehends providing atreatment or delaying the onset or progression of Leber's CongenitalAmaurosis 10 (LCA 10). LCA 10 is caused by a mutation in the CEP290gene, e.g., a c.2991+1655, adenine to guanine mutation in the CEP290gene which gives rise to a cryptic splice site in intron 26. This is amutation at nucleotide 1655 of intron 26 of CEP290, e.g., an A to Gmutation. CEP290 is also known as: CT87; MKS4; POC3; rd16; BBS14; JBTS5;LCAJO; NPHP6; SLSN6; and 3H11Ag (see, e.g., WO 2015/138510). In anaspect of gene therapy, the invention involves introducing one or morebreaks near the site of the LCA target position (e.g., c.2991+1655; A toG) in at least one allele of the CEP290 gene. Altering the LCA10 targetposition refers to (1) break-induced introduction of an indel (alsoreferred to herein as NHEJ-mediated introduction of an indel) in closeproximity to or including a LCA10 target position (e.g., c.2991+1655A toG), or (2) break-induced deletion (also referred to herein asNHEJ-mediated deletion) of genomic sequence including the mutation at aLCA10 target position (e.g., c.2991+1655A to G). Both approaches giverise to the loss or destruction of the cryptic splice site resultingfrom the mutation at the LCA 10 target position. Accordingly, the use ofCpf1 in the treatment of LCA is specifically envisaged.

Researchers are contemplating whether gene therapies could be employedto treat a wide range of diseases. The CRISPR systems of the presentinvention based on Cpf1 effector protein are envisioned for suchtherapeutic uses, including, but noted limited to further exemplifiedtargeted areas and with delivery methods as below. Some examples ofconditions or diseases that might be usefully treated using the presentsystem are included in the examples of genes and references includedherein and are currently associated with those conditions are alsoprovided there. The genes and conditions exemplified are not exhaustive.

Treating Diseases of the Circulatory System

The present invention also contemplates delivering the CRISPR-Cassystem, specifically the novel CRISPR effector protein systems describedherein, to the blood or hematopoetic stem cells. The plasma exosomes ofWahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130)were previously described and may be utilized to deliver the CRISPR Cassystem to the blood. The nucleic acid-targeting system of the presentinvention is also contemplated to treat hemoglobinopathies, such asthalassemias and sickle cell disease. See, e.g., International PatentPublication No. WO 2013/126794 for potential targets that may betargeted by the CRISPR Cas system of the present invention.

Drakopoulou, “Review Article, The Ongoing Challenge of HematopoieticStem Cell-Based Gene Therapy for β-Thalassemia,” Stem CellsInternational, Volume 2011, Article ID 987980, 10 pages,doi:10.4061/2011/987980, incorporated herein by reference along with thedocuments it cites, as if set out in full, discuss modifying HSCs usinga lentivirus that delivers a gene for β-globin or γ-globin. In contrastto using lentivirus, with the knowledge in the art and the teachings inthis disclosure, the skilled person can correct HSCs as to β-Thalassemiausing a CRISPR-Cas system that targets and corrects the mutation (e.g.,with a suitable HDR template that delivers a coding sequence forβ-globin or γ-globin, advantageously non-sickling β-globin or γ-globin);specifically, the guide RNA can target mutation that give rise toβ-Thalassemia, and the HDR can provide coding for proper expression ofβ-globin or γ-globin. An guide RNA that targets the mutation-and-Casprotein containing particle is contacted with HSCs carrying themutation. The particle also can contain a suitable HDR template tocorrect the mutation for proper expression of β-globin or γ-globin; orthe HSC can be contacted with a second particle or a vector thatcontains or delivers the HDR template. The so contacted cells can beadministered; and optionally treated/expanded; cf. Cartier. In thisregard mention is made of: Cavazzana, “Outcomes of Gene Therapy forβ-Thalassemia Major via Transplantation of Autologous Hematopoietic StemCells Transduced Ex Vivo with a Lentiviral β^(A-T87Q)-Globin Vector.”tif2014.org/abstractFiles/Jean%20Antoine%20Ribeil_Abstract.pdf;Cavazzana-Calvo, “Transfusion independence and HMGA2 activation aftergene therapy of human β-thalassaemia”, Nature 467, 318-322 (16 Sep.2010) doi:10.1038/nature09328; Nienhuis, “Development of Gene Therapyfor Thalassemia, Cold Spring Harbor Perpsectives in Medicine, doi:10.1101/cshperspect.a011833 (2012), LentiGlobin BB305, a lentiviralvector containing an engineered β-globin gene (βA-T87Q); and Xie et al.,“Seamless gene correction of β-thalassaemia mutations inpatient-specific iPSCs using CRISPR/Cas9 and piggyback” Genome Researchgr.173427.114 (2014) http://www.genome.org/cgi/doi/10.1101/gr.173427.114(Cold Spring Harbor Laboratory Press); that is the subject of Cavazzanawork involving human β-thalassaemia and the subject of the Xie work, areall incorporated herein by reference, together with all documents citedtherein or associated therewith. In the instant invention, the HDRtemplate can provide for the HSC to express an engineered β-globin gene(e.g., βA-T87Q), or β-globin as in Xie.

Xu et al. (Sci Rep. 2015 Jul. 9; 5:12065. doi: 10.1038/srep12065) havedesigned TALENs and CRISPR-Cas9 to directly target the intron2 mutationsite IVS2-654 in the globin gene. Xu et al. observed differentfrequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENsand CRISPR-Cas9, and TALENs mediated a higher homologous gene targetingefficiency compared to CRISPR-Cas9 when combined with the piggyBactransposon donor. In addition, more obvious off-target events wereobserved for CRISPR-Cas9 compared to TALENs. Finally, TALENs-correctediPSC clones were selected for erythroblast differentiation using the OP9co-culture system and detected relatively higher transcription of HBBthan the uncorrected cells.

Song et al. (Stem Cells Dev. 2015 May 1; 24(9):1053-65. doi:10.1089/scd.2014.0347. Epub 2015 Feb. 5) used CRISPR/Cas9 to correctβ-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and fullpluripotency as human embryonic stem cells (hESCs) showed nooff-targeting effects. Then, Song et al. evaluated the differentiationefficiency of the gene-corrected β-Thal iPSCs. Song et al. found thatduring hematopoietic differentiation, gene-corrected β-Thal iPSCs showedan increased embryoid body ratio and various hematopoietic progenitorcell percentages. More importantly, the gene-corrected β-Thal iPSC linesrestored HBB expression and reduced reactive oxygen species productioncompared with the uncorrected group. Song et al.'s study suggested thathematopoietic differentiation efficiency of β-Thal iPSCs was greatlyimproved once corrected by the CRISPR-Cas9 system. Similar methods maybe performed utilizing the CRISPR-Cas systems described herein, e.g.systems comprising Cpf1 effector proteins.

Sickle cell anemia is an autosomal recessive genetic disease in whichred blood cells become sickle-shaped. It is caused by a single basesubstitution in the β-globin gene, which is located on the short arm ofchromosome 11. As a result, valine is produced instead of glutamic acidcausing the production of sickle hemoglobin (HbS). This results in theformation of a distorted shape of the erythrocytes. Due to this abnormalshape, small blood vessels can be blocked, causing serious damage to thebone, spleen and skin tissues. This may lead to episodes of pain,frequent infections, hand-foot syndrome or even multiple organ failure.The distorted erythrocytes are also more susceptible to hemolysis, whichleads to serious anemia. As in the case of β-thalassaemia, sickle cellanemia can be corrected by modifying HSCs with the CRISPR-Cas system.The system allows the specific editing of the cell's genome by cuttingits DNA and then letting it repair itself. The Cas protein is insertedand directed by a RNA guide to the mutated point and then it cuts theDNA at that point. Simultaneously, a healthy version of the sequence isinserted. This sequence is used by the cell's own repair system to fixthe induced cut. In this way, the CRISPR-Cas allows the correction ofthe mutation in the previously obtained stem cells. With the knowledgein the art and the teachings in this disclosure, the skilled person cancorrect HSCs as to sickle cell anemia using a CRISPR-Cas system thattargets and corrects the mutation (e.g., with a suitable HDR templatethat delivers a coding sequence for β-globin, advantageouslynon-sickling β-globin); specifically, the guide RNA can target mutationthat give rise to sickle cell anemia, and the HDR can provide coding forproper expression of β-globin. An guide RNA that targets themutation-and-Cas protein containing particle is contacted with HSCscarrying the mutation. The particle also can contain a suitable HDRtemplate to correct the mutation for proper expression of β-globin; orthe HSC can be contacted with a second particle or a vector thatcontains or delivers the HDR template. The so contacted cells can beadministered; and optionally treated/expanded; cf. Cartier. The HDRtemplate can provide for the HSC to express an engineered β-globin gene(e.g., βA-T87Q), or β-globin as in Xie.

Williams, “Broadening the Indications for Hematopoietic Stem CellGenetic Therapies,” Cell Stem Cell 13:263-264 (2013), incorporatedherein by reference along with the documents it cites, as if set out infull, report lentivirus-mediated gene transfer into HSC/P cells frompatients with the lysosomal storage disease metachromatic leukodystrophydisease (MLD), a genetic disease caused by deficiency of arylsulfatase A(ARSA), resulting in nerve demyelination; and lentivirus-mediated genetransfer into HSCs of patients with Wiskott-Aldrich syndrome (WAS)(patients with defective WAS protein, an effector of the small GTPaseCDCl42 that regulates cytoskeletal function in blood cell lineages andthus suffer from immune deficiency with recurrent infections, autoimmunesymptoms, and thrombocytopenia with abnormally small and dysfunctionalplatelets leading to excessive bleeding and an increased risk ofleukemia and lymphoma). In contrast to using lentivirus, with theknowledge in the art and the teachings in this disclosure, the skilledperson can correct HSCs as to MLD (deficiency of arylsulfatase A (ARSA))using a CRISPR-Cas system that targets and corrects the mutation(deficiency of arylsulfatase A (ARSA)) (e.g., with a suitable HDRtemplate that delivers a coding sequence for ARSA); specifically, theguide RNA can target mutation that gives rise to MLD (deficient ARSA),and the HDR can provide coding for proper expression of ARSA. An guideRNA that targets the mutation-and-Cas protein containing particle iscontacted with HSCs carrying the mutation. The particle also can containa suitable HDR template to correct the mutation for proper expression ofARSA; or the HSC can be contacted with a second particle or a vectorthat contains or delivers the HDR template. The so contacted cells canbe administered; and optionally treated/expanded; cf. Cartier. Incontrast to using lentivirus, with the knowledge in the art and theteachings in this disclosure, the skilled person can correct HSCs as toWAS using a CRISPR-Cas system that targets and corrects the mutation(deficiency of WAS protein) (e.g., with a suitable HDR template thatdelivers a coding sequence for WAS protein); specifically, the guide RNAcan target mutation that gives rise to WAS (deficient WAS protein), andthe HDR can provide coding for proper expression of WAS protein. Anguide RNA that targets the mutation-and-Cpf1 protein containing particleis contacted with HSCs carrying the mutation. The particle also cancontain a suitable HDR template to correct the mutation for properexpression of WAS protein; or the HSC can be contacted with a secondparticle or a vector that contains or delivers the HDR template. The socontacted cells can be administered; and optionally treated/expanded;cf. Cartier.

Watts, “Hematopoietic Stem Cell Expansion and Gene Therapy” Cytotherapy13(10):1164-1171. doi:10.3109/14653249.2011.620748 (2011), incorporatedherein by reference along with the documents it cites, as if set out infull, discusses hematopoietic stem cell (HSC) gene therapy, e.g.,virus-mediated HSC gene therapy, as an highly attractive treatmentoption for many disorders including hematologic conditions,immunodeficiencies including HIV/AIDS, and other genetic disorders likelysosomal storage diseases, including SCID-X1, ADA-SCID, β-thalassemia,X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia,adrenoleukodystrophy (ALD), and metachromatic leukodystrophy (MLD).

US Patent Publication Nos. 20110225664, 20110091441, 20100229252,20090271881 and 20090222937 assigned to Cellectis, relates to CREIvariants, wherein at least one of the two I-CreI monomers has at leasttwo substitutions, one in each of the two functional subdomains of theLAGLIDADG (SEQ ID NO: 26) core domain situated respectively frompositions 26 to 40 and 44 to 77 of I-CreI, said variant being able tocleave a DNA target sequence from the human interleukin-2 receptor gammachain (IL2RG) gene also named common cytokine receptor gamma chain geneor gamma C gene. The target sequences identified in US PatentPublication Nos. 20110225664, 20110091441, 20100229252, 20090271881 and20090222937 may be utilized for the nucleic acid-targeting system of thepresent invention.

Severe Combined Immune Deficiency (SCID) results from a defect inlymphocytes T maturation, always associated with a functional defect inlymphocytes B (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56,585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109). Overallincidence is estimated to 1 in 75 000 births. Patients with untreatedSCID are subject to multiple opportunist micro-organism infections, anddo generally not live beyond one year. SCID can be treated by allogenichematopoietic stem cell transfer, from a familial donor.Histocompatibility with the donor can vary widely. In the case ofAdenosine Deaminase (ADA) deficiency, one of the SCID forms, patientscan be treated by injection of recombinant Adenosine Deaminase enzyme.

Since the ADA gene has been shown to be mutated in SCID patients(Giblett et al., Lancet, 1972, 2, 1067-1069), several other genesinvolved in SCID have been identified (Cavazzana-Calvo et al., Annu.Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203,98-109). There are four major causes for SCID: (i) the most frequentform of SCID, SCID-X1 (X-linked SCID or X-SCID), is caused by mutationin the IL2RG gene, resulting in the absence of mature T lymphocytes andNK cells. IL2RG encodes the gamma C protein (Noguchi, et al., Cell,1993, 73, 147-157), a common component of at least five interleukinreceptor complexes. These receptors activate several targets through theJAK3 kinase (Macchi et al., Nature, 1995, 377, 65-68), whichinactivation results in the same syndrome as gamma C inactivation; (ii)mutation in the ADA gene results in a defect in purine metabolism thatis lethal for lymphocyte precursors, which in turn results in the quasiabsence of B, T and NK cells; (iii) V(D)J recombination is an essentialstep in the maturation of immunoglobulins and T lymphocytes receptors(TCRs). Mutations in Recombination Activating Gene 1 and 2 (RAG1 andRAG2) and Artemis, three genes involved in this process, result in theabsence of mature T and B lymphocytes; and (iv) Mutations in other genessuch as CD45, involved in T cell specific signaling have also beenreported, although they represent a minority of cases (Cavazzana-Calvoet al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol.Rev., 2005, 203, 98-109). Since when their genetic bases have beenidentified, the different SCID forms have become a paradigm for genetherapy approaches (Fischer et al., Immunol. Rev., 2005, 203, 98-109)for two major reasons. First, as in all blood diseases, an ex vivotreatment can be envisioned. Hematopoietic Stem Cells (HSCs) can berecovered from bone marrow, and keep their pluripotent properties for afew cell divisions. Therefore, they can be treated in vitro, and thenreinjected into the patient, where they repopulate the bone marrow.Second, since the maturation of lymphocytes is impaired in SCIDpatients, corrected cells have a selective advantage. Therefore, a smallnumber of corrected cells can restore a functional immune system. Thishypothesis was validated several times by (i) the partial restoration ofimmune functions associated with the reversion of mutations in SCIDpatients (Hirschhorn et al., Nat. Genet., 1996, 13, 290-295; Stephan etal., N. Engl. J. Med., 1996, 335, 1563-1567; Bousso et al., Proc. Natl.,Acad. Sci. USA, 2000, 97, 274-278; Wada et al., Proc. Natl. Acad. Sci.USA, 2001, 98, 8697-8702; Nishikomori et al., Blood, 2004, 103,4565-4572), (ii) the correction of SCID-X1 deficiencies in vitro inhematopoietic cells (Candotti et al., Blood, 1996, 87, 3097-3102;Cavazzana-Calvo et al., Blood, 1996, Blood, 88, 3901-3909; Taylor etal., Blood, 1996, 87, 3103-3107; Hacein-Bey et al., Blood, 1998, 92,4090-4097), (iii) the correction of SCID-X1 (Soudais et al., Blood,2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JAK-3(Bunting et al., Nat. Med., 1998, 4, 58-64; Bunting et al., Hum. GeneTher., 2000, 11, 2353-2364) and RAG2 (Yates et al., Blood, 2002, 100,3942-3949) deficiencies in vivo in animal models and (iv) by the resultof gene therapy clinical trials (Cavazzana-Calvo et al., Science, 2000,288, 669-672; Aiuti et al., Nat. Med., 2002; 8, 423-425; Gaspar et al.,Lancet, 2004, 364, 2181-2187).

US Patent Publication No. 20110182867 assigned to the Children's MedicalCenter Corporation and the President and Fellows of Harvard Collegerelates to methods and uses of modulating fetal hemoglobin expression(HbF) in a hematopoietic progenitor cells via inhibitors of BCL11Aexpression or activity, such as RNAi and antibodies. The targetsdisclosed in US Patent Publication No. 20110182867, such as BCL11A, maybe targeted by the CRISPR Cas system of the present invention formodulating fetal hemoglobin expression. See also Bauer et al. (Science11 Oct. 2013: Vol. 342 no. 6155 pp. 253-257) and Xu et al. (Science 18Nov. 2011: Vol. 334 no. 6058 pp. 993-996) for additional BCL11A targets.

With the knowledge in the art and the teachings in this disclosure, theskilled person can correct HSCs as to a genetic hematologic disorder,e.g., β-Thalassemia, Hemophilia, or a genetic lysosomal storage disease.

HSC—Delivery to and Editing of Hematopoetic Stem Cells; and ParticularConditions.

The term “Hematopoetic Stem Cell” or “HSC” is meant to include broadlythose cells considered to be an HSC, e.g., blood cells that give rise toall the other blood cells and are derived from mesoderm; located in thered bone marrow, which is contained in the core of most bones. HSCs ofthe invention include cells having a phenotype of hematopoeitic stemcells, identified by small size, lack of lineage (lin) markers, andmarkers that belong to the cluster of differentiation series, like:CD34, CD38, CD90, CD133, CD105, CD45, and also c-kit, —the receptor forstem cell factor. Hematopoietic stem cells are negative for the markersthat are used for detection of lineage commitment, and are, thus, calledLin-; and, during their purification by FACS, a number of up to 14different mature blood-lineage markers, e.g., CD13 & CD33 for myeloid,CD71 for erythroid, CD19 for B cells, CD61 for megakaryocytic, etc. forhumans; and, B220 (murine CD45) for B cells, Mac-1 (CD11b/CD18) formonocytes, Gr-1 for Granulocytes, Ter119 for erythroid cells, Il7Ra,CD3, CD4, CDS, CD8 for T cells, etc. Mouse HSC markers: CD341o/−,SCA-1+, Thy1.1+/lo, CD38+, C-kit+, lin−, and Human HSC markers: CD34+,CD59+, Thy1/CD90+, CD381o/−, C-kit/CD117+, and lin−. HSCs are identifiedby markers. Hence in embodiments discussed herein, the HSCs can be CD34+cells. HSCs can also be hematopoietic stem cells that are CD34−/CD38−.Stem cells that may lack c-kit on the cell surface that are consideredin the art as HSCs are within the ambit of the invention, as well asCD133+ cells likewise considered HSCs in the art.

The CRISPR-Cas (eg Cpf1) system may be engineered to target geneticlocus or loci in HSCs. Cas (eg Cpf1) protein, advantageouslycodon-optimized for a eukaryotic cell and especially a mammalian cell,e.g., a human cell, for instance, HSC, and sgRNA targeting a locus orloci in HSC, e.g., the gene EMX1, may be prepared. These may bedelivered via particles. The particles may be formed by the Cas (egCpf1) protein and the gRNA being admixed. The gRNA and Cas (eg Cpf1)protein mixture may for example be admixed with a mixture comprising orconsisting essentially of or consisting of surfactant, phospholipid,biodegradable polymer, lipoprotein and alcohol, whereby particlescontaining the gRNA and Cas (eg Cpf1) protein may be formed. Theinvention comprehends so making particles and particles from such amethod as well as uses thereof.

More generally, particles may be formed using an efficient process.First, Cas (eg Cpf1) protein and gRNA targeting the gene EMX1 or thecontrol gene LacZ may be mixed together at a suitable, e.g., 3:1 to 1:3or 2:1 to 1:2 or 1:1 molar ratio, at a suitable temperature, e.g., 15-30C, e.g., 20-25 C, e.g., room temperature, for a suitable time, e.g.,15-45, such as 30 minutes, advantageously in sterile, nuclease freebuffer, e.g., 1×PBS. Separately, particle components such as orcomprising: a surfactant, e.g., cationic lipid, e.g.,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); phospholipid, e.g.,dimyristoylphosphatidylcholine (DMPC); biodegradable polymer, such as anethylene-glycol polymer or PEG, and a lipoprotein, such as a low-densitylipoprotein, e.g., cholesterol may be dissolved in an alcohol,advantageously a C1-6 alkyl alcohol, such as methanol, ethanol,isopropanol, e.g., 100% ethanol. The two solutions may be mixed togetherto form particles containing the Cas (eg Cpf1)-gRNA complexes. Incertain embodiments the particle can contain an HDR template. That canbe a particle co-administered with gRNA+Cas (eg Cpf1) protein-containingparticle, or i.e., in addition to contacting an HSC with an gRNA+Cas (egCpf1) protein-containing particle, the HSC is contacted with a particlecontaining an HDR template; or the HSC is contacted with a particlecontaining all of the gRNA, Cas (eg Cpf1) and the HDR template. The HDRtemplate can be administered by a separate vector, whereby in a firstinstance the particle penetrates an HSC cell and the separate vectoralso penetrates the cell, wherein the HSC genome is modified by thegRNA+Cas (eg Cpf1) and the HDR template is also present, whereby agenomic loci is modified by the HDR; for instance, this may result incorrecting a mutation.

After the particles form, HSCs in 96 well plates may be transfected with15 ug Cas (eg Cpf1) protein per well. Three days after transfection,HSCs may be harvested, and the number of insertions and deletions(indels) at the EMX1 locus may be quantified.

This illustrates how HSCs can be modified using CRISPR-Cas (eg Cpf1)targeting a genomic locus or loci of interest in the HSC. The HSCs thatare to be modified can be in vivo, i.e., in an organism, for example ahuman or a non-human eukaryote, e.g., animal, such as fish, e.g., zebrafish, mammal, e.g., primate, e.g., ape, chimpanzee, macaque, rodent,e.g., mouse, rabbit, rat, canine or dog, livestock (cow/bovine,sheep/ovine, goat or pig), fowl or poultry, e.g., chicken. The HSCs thatare to be modified can be in vitro, i.e., outside of such an organism.And, modified HSCs can be used ex vivo, i.e., one or more HSCs of suchan organism can be obtained or isolated from the organism, optionallythe HSC(s) can be expanded, the HSC(s) are modified by a compositioncomprising a CRISPR-Cas (eg Cpf1) that targets a genetic locus or lociin the HSC, e.g., by contacting the HSC(s) with the composition, forinstance, wherein the composition comprises a particle containing theCRISPR enzyme and one or more gRNA that targets the genetic locus orloci in the HSC, such as a particle obtained or obtainable from admixingan gRNA and Cas (eg Cpf1) protein mixture with a mixture comprising orconsisting essentially of or consisting of surfactant, phospholipid,biodegradable polymer, lipoprotein and alcohol (wherein one or more gRNAtargets the genetic locus or loci in the HSC), optionally expanding theresultant modified HSCs and administering to the organism the resultantmodified HSCs. In some instances the isolated or obtained HSCs can befrom a first organism, such as an organism from a same species as asecond organism, and the second organism can be the organism to whichthe resultant modified HSCs are administered, e.g., the first organismcan be a donor (such as a relative as in a parent or sibling) to thesecond organism. Modified HSCs can have genetic modifications to addressor alleviate or reduce symptoms of a disease or condition state of anindividual or subject or patient. Modified HSCs, e.g., in the instanceof a first organism donor to a second organism, can have geneticmodifications to have the HSCs have one or more proteins e.g. surfacemarkers or proteins more like that of the second organism. Modified HSCscan have genetic modifications to simulate a disease or condition stateof an individual or subject or patient and would be re-administered to anon-human organism so as to prepare an animal model. Expansion of HSCsis within the ambit of the skilled person from this disclosure andknowledge in the art, see e.g., Lee, “Improved ex vivo expansion ofadult hematopoietic stem cells by overcoming CUL4-mediated degradationof HOXB4.” Blood. 2013 May 16; 121(20):4082-9. doi:10.1182/blood-2012-09-455204. Epub 2013 Mar. 21.

As indicated to improve activity, gRNA may be pre-complexed with the Cas(eg Cpf1) protein, before formulating the entire complex in a particle.Formulations may be made with a different molar ratio of differentcomponents known to promote delivery of nucleic acids into cells (e.g.1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP:DMPC:PEG:CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. The inventionaccordingly comprehends admixing gRNA, Cas (eg Cpf1) protein andcomponents that form a particle; as well as particles from suchadmixing.

In a preferred embodiment, particles containing the Cas (eg Cpf1)-gRNAcomplexes may be formed by mixing Cas (eg Cpf1) protein and one or moregRNAs together, preferably at a 1:1 molar ratio, enzyme:guide RNA.Separately, the different components known to promote delivery ofnucleic acids (e.g. DOTAP, DMPC, PEG, and cholesterol) are dissolved,preferably in ethanol. The two solutions are mixed together to formparticles containing the Cas (eg Cpf1)-gRNA complexes. After theparticles are formed, Cas (eg Cpf1)-gRNA complexes may be transfectedinto cells (e.g. HSCs). Bar coding may be applied. The particles, theCas-9 and/or the gRNA may be barcoded.

The invention in an embodiment comprehends a method of preparing angRNA-and-Cas (eg Cpf1) protein containing particle comprising admixingan gRNA and Cas (eg Cpf1) protein mixture with a mixture comprising orconsisting essentially of or consisting of surfactant, phospholipid,biodegradable polymer, lipoprotein and alcohol. An embodimentcomprehends an gRNA-and-Cas (eg Cpf1) protein containing particle fromthe method. The invention in an embodiment comprehends use of theparticle in a method of modifying a genomic locus of interest, or anorganism or a non-human organism by manipulation of a target sequence ina genomic locus of interest, comprising contacting a cell containing thegenomic locus of interest with the particle wherein the gRNA targets thegenomic locus of interest; or a method of modifying a genomic locus ofinterest, or an organism or a non-human organism by manipulation of atarget sequence in a genomic locus of interest, comprising contacting acell containing the genomic locus of interest with the particle whereinthe gRNA targets the genomic locus of interest. In these embodiments,the genomic locus of interest is advantageously a genomic locus in anHSC.

Considerations for Therapeutic Applications: A consideration in genomeediting therapy is the choice of sequence-specific nuclease, such as avariant of a Cpf1 nuclease. Each nuclease variant may possess its ownunique set of strengths and weaknesses, many of which must be balancedin the context of treatment to maximize therapeutic benefit. Thus far,two therapeutic editing approaches with nucleases have shown significantpromise: gene disruption and gene correction. Gene disruption involvesstimulation of NHEJ to create targeted indels in genetic elements, oftenresulting in loss of function mutations that are beneficial to patients.In contrast, gene correction uses HDR to directly reverse a diseasecausing mutation, restoring function while preserving physiologicalregulation of the corrected element. HDR may also be used to insert atherapeutic transgene into a defined ‘safe harbor’ locus in the genometo recover missing gene function. For a specific editing therapy to beefficacious, a sufficiently high level of modification must be achievedin target cell populations to reverse disease symptoms. This therapeuticmodification ‘threshold’ is determined by the fitness of edited cellsfollowing treatment and the amount of gene product necessary to reversesymptoms. With regard to fitness, editing creates three potentialoutcomes for treated cells relative to their unedited counterparts:increased, neutral, or decreased fitness. In the case of increasedfitness, for example in the treatment of SCID-X1, modified hematopoieticprogenitor cells selectively expand relative to their uneditedcounterparts. SCID-X1 is a disease caused by mutations in the IL2RGgene, the function of which is required for proper development of thehematopoietic lymphocyte lineage [Leonard, W. J., et al. Immunologicalreviews 138, 61-86 (1994); Kaushansky, K. & Williams, W. J. Williamshematology, (McGraw-Hill Medical, New York, 2010)]. In clinical trialswith patients who received viral gene therapy for SCID-X1, and a rareexample of a spontaneous correction of SCID-X1 mutation, correctedhematopoietic progenitor cells may be able to overcome thisdevelopmental block and expand relative to their diseased counterpartsto mediate therapy [Bousso, P., et al. Proceedings of the NationalAcademy of Sciences of the United States of America 97, 274-278 (2000);Hacein-Bey-Abina, S., et al. The New England journal of medicine 346,1185-1193 (2002); Gaspar, H. B., et al. Lancet 364, 2181-2187 (2004)].In this case, where edited cells possess a selective advantage, even lownumbers of edited cells can be amplified through expansion, providing atherapeutic benefit to the patient. In contrast, editing for otherhematopoietic diseases, like chronic granulomatous disorder (CGD), wouldinduce no change in fitness for edited hematopoietic progenitor cells,increasing the therapeutic modification threshold. CGD is caused bymutations in genes encoding phagocytic oxidase proteins, which arenormally used by neutrophils to generate reactive oxygen species thatkill pathogens [Mukherjee, S. & Thrasher, A. J. Gene 525, 174-181(2013)]. As dysfunction of these genes does not influence hematopoieticprogenitor cell fitness or development, but only the ability of a maturehematopoietic cell type to fight infections, there would be likely nopreferential expansion of edited cells in this disease. Indeed, noselective advantage for gene corrected cells in CGD has been observed ingene therapy trials, leading to difficulties with long-term cellengraftment [Malech, H. L., et al. Proceedings of the National Academyof Sciences of the United States of America 94, 12133-12138 (1997);Kang, H. J., et al. Molecular therapy: the journal of the AmericanSociety of Gene Therapy 19, 2092-2101 (2011)]. As such, significantlyhigher levels of editing would be required to treat diseases like CGD,where editing creates a neutral fitness advantage, relative to diseaseswhere editing creates increased fitness for target cells. If editingimposes a fitness disadvantage, as would be the case for restoringfunction to a tumor suppressor gene in cancer cells, modified cellswould be outcompeted by their diseased counterparts, causing the benefitof treatment to be low relative to editing rates. This latter class ofdiseases would be particularly difficult to treat with genome editingtherapy.

In addition to cell fitness, the amount of gene product necessary totreat disease also influences the minimal level of therapeutic genomeediting that must be achieved to reverse symptoms. Haemophilia B is onedisease where a small change in gene product levels can result insignificant changes in clinical outcomes. This disease is caused bymutations in the gene encoding factor IX, a protein normally secreted bythe liver into the blood, where it functions as a component of theclotting cascade. Clinical severity of haemophilia B is related to theamount of factor IX activity. Whereas severe disease is associated withless than 1% of normal activity, milder forms of the diseases areassociated with greater than 1% of factor IX activity [Kaushansky, K. &Williams, W. J. Williams hematology, (McGraw-Hill Medical, New York,2010); Lofqvist, T., et al. Journal of internal medicine 241, 395-400(1997)]. This suggests that editing therapies that can restore factor IXexpression to even a small percentage of liver cells could have a largeimpact on clinical outcomes. A study using ZFNs to correct a mouse modelof haemophilia B shortly after birth demonstrated that 3-7% correctionwas sufficient to reverse disease symptoms, providing preclinicalevidence for this hypothesis [Li, H., et al. Nature 475, 217-221(2011)].

Disorders where a small change in gene product levels can influenceclinical outcomes and diseases where there is a fitness advantage foredited cells, are ideal targets for genome editing therapy, as thetherapeutic modification threshold is low enough to permit a high chanceof success given the current technology. Targeting these diseases hasnow resulted in successes with editing therapy at the preclinical leveland a phase I clinical trial. Improvements in DSB repair pathwaymanipulation and nuclease delivery are needed to extend these promisingresults to diseases with a neutral fitness advantage for edited cells,or where larger amounts of gene product are needed for treatment. TheTable below shows some examples of applications of genome editing totherapeutic models, and the references of the below Table and thedocuments cited in those references are hereby incorporated herein byreference as if set out in full.

Nuclease Disease Platform Type Employed Therapeutic Strategy ReferencesHemophilia ZFN HDR-mediated Li, H., et al. Nature B insertion of correct475, 217-221 (2011) gene sequence SCID ZFN HDR-mediated Genovese, P., etal. insertion of correct Nature 510, 235-240 gene sequence (2014)Hereditary CRISPR HDR-mediated Yin, H., et al. Nature tyrosinemiacorrection of mutation biotechnology 32, in liver 551-553 (2014)

Addressing each of the conditions of the foregoing table, using theCRISPR-Cas (eg Cpf1) system to target by either HDR-mediated correctionof mutation, or HDR-mediated insertion of correct gene sequence,advantageously via a delivery system as herein, e.g., a particledelivery system, is within the ambit of the skilled person from thisdisclosure and the knowledge in the art. Thus, an embodiment comprehendscontacting a Hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or Hereditarytyrosinemia mutation-carrying HSC with an gRNA-and-Cas (eg Cpf1) proteincontaining particle targeting a genomic locus of interest as toHemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or Hereditary tyrosinemia(e.g., as in Li, Genovese or Yin). The particle also can contain asuitable HDR template to correct the mutation; or the HSC can becontacted with a second particle or a vector that contains or deliversthe HDR template. In this regard, it is mentioned that Haemophilia B isan X-linked recessive disorder caused by loss-of-function mutations inthe gene encoding Factor IX, a crucial component of the clottingcascade. Recovering Factor IX activity to above 1% of its levels inseverely affected individuals can transform the disease into asignificantly milder form, as infusion of recombinant Factor IX intosuch patients prophylactically from a young age to achieve such levelslargely ameliorates clinical complications. With the knowledge in theart and the teachings in this disclosure, the skilled person can correctHSCs as to Haemophilia B using a CRISPR-Cas (eg Cpf1) system thattargets and corrects the mutation (X-linked recessive disorder caused byloss-of-function mutations in the gene encoding Factor IX) (e.g., with asuitable HDR template that delivers a coding sequence for Factor IX);specifically, the gRNA can target mutation that give rise to HaemophiliaB, and the HDR can provide coding for proper expression of Factor IX. AngRNA that targets the mutation-and-Cas (eg Cpf1) protein containingparticle is contacted with HSCs carrying the mutation. The particle alsocan contain a suitable HDR template to correct the mutation for properexpression of Factor IX; or the HSC can be contacted with a secondparticle or a vector that contains or delivers the HDR template. The socontacted cells can be administered; and optionally treated/expanded;cf. Cartier, discussed herein.

In Cartier, “MINI-SYMPOSIUM: X-Linked Adrenoleukodystrophypa,Hematopoietic Stem Cell Transplantation and Hematopoietic Stem Cell GeneTherapy in X-Linked Adrenoleukodystrophy,” Brain Pathology 20 (2010)857-862, incorporated herein by reference along with the documents itcites, as if set out in full, there is recognition that allogeneichematopoietic stem cell transplantation (HSCT) was utilized to delivernormal lysosomal enzyme to the brain of a patient with Hurler's disease,and a discussion of HSC gene therapy to treat ALD. In two patients,peripheral CD34+ cells were collected after granulocyte-colonystimulating factor (G-CSF) mobilization and transduced with anmyeloproliferative sarcoma virus enhancer, negative control regiondeleted, d1587rev primer binding site substituted (MND)-ALD lentiviralvector. CD34+ cells from the patients were transduced with the MND-ALDvector during 16 h in the presence of cytokines at low concentrations.Transduced CD34+ cells were frozen after transduction to perform on 5%of cells various safety tests that included in particular threereplication-competent lentivirus (RCL) assays. Transduction efficacy ofCD34+ cells ranged from 35% to 50% with a mean number of lentiviralintegrated copy between 0.65 and 0.70. After the thawing of transducedCD34+ cells, the patients were reinfused with more than 4.106 transducedCD34+ cells/kg following full myeloablation with busulfan andcyclophos-phamide. The patient's HSCs were ablated to favor engraftmentof the gene-corrected HSCs. Hematological recovery occurred between days13 and 15 for the two patients. Nearly complete immunological recoveryoccurred at 12 months for the first patient, and at 9 months for thesecond patient. In contrast to using lentivirus, with the knowledge inthe art and the teachings in this disclosure, the skilled person cancorrect HSCs as to ALD using a CRISPR-Cas (Cpf1) system that targets andcorrects the mutation (e.g., with a suitable HDR template);specifically, the gRNA can target mutations in ABCD1, a gene located onthe X chromosome that codes for ALD, a peroxisomal membrane transporterprotein, and the HDR can provide coding for proper expression of theprotein. An gRNA that targets the mutation-and-Cas (Cpf1) proteincontaining particle is contacted with HSCs, e.g., CD34+ cells carryingthe mutation as in Cartier. The particle also can contain a suitable HDRtemplate to correct the mutation for expression of the peroxisomalmembrane transporter protein; or the HSC can be contacted with a secondparticle or a vector that contains or delivers the HDR template. The socontacted cells optionally can be treated as in Cartier. The socontacted cells can be administered as in Cartier.

Mention is made of WO 2015/148860, through the teachings herein theinvention comprehends methods and materials of these documents appliedin conjunction with the teachings herein. In an aspect of blood-relateddisease gene therapy, methods and compositions for treating betathalassemia may be adapted to the CRISPR-Cas system of the presentinvention (see, e.g., WO 2015/148860). In an embodiment, WO 2015/148860involves the treatment or prevention of beta thalassemia, or itssymptoms, e.g., by altering the gene for B-cell CLL/lymphoma 11A(BCL11A). The BCL11A gene is also known as B-cell CLL/lymphoma 11A,BCL11A-L, BCL11A-S, BCL11AXL, CTIP 1, HBFQTLS and ZNF. BCL11A encodes azinc-finger protein that is involved in the regulation of globin geneexpression. By altering the BCL11A gene (e.g., one or both alleles ofthe BCL11A gene), the levels of gamma globin can be increased. Gammaglobin can replace beta globin in the hemoglobin complex and effectivelycarry oxygen to tissues, thereby ameliorating beta thalassemia diseasephenotypes.

Mention is also made of WO 2015/148863 and through the teachings hereinthe invention comprehends methods and materials of these documents whichmay be adapted to the CRISPR-Cas system of the present invention. In anaspect of treating and preventing sickle cell disease, which is aninherited hematologic disease, WO 2015/148863 comprehends altering theBCL11A gene. By altering the BCL11A gene (e.g., one or both alleles ofthe BCL11A gene), the levels of gamma globin can be increased. Gammaglobin can replace beta globin in the hemoglobin complex and effectivelycarry oxygen to tissues, thereby ameliorating sickle cell diseasephenotypes.

In an aspect of the invention, methods and compositions which involveediting a target nucleic acid sequence, or modulating expression of atarget nucleic acid sequence, and applications thereof in connectionwith cancer immunotherapy are comprehended by adapting the CRISPR-Cassystem of the present invention. Reference is made to the application ofgene therapy in WO 2015/161276 which involves methods and compositionswhich can be used to affect T-cell proliferation, survival and/orfunction by altering one or more T-cell expressed genes, e.g., one ormore of FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC and/or TRBC genes. Ina related aspect, T-cell proliferation can be affected by altering oneor more T-cell expressed genes, e.g., the CBLB and/or PTPN6 gene, FASand/or BID gene, CTLA4 and/or PDCDI and/or TRAC and/or TRBC gene.

Chimeric antigen receptor (CAR)19 T-cells exhibit anti-leukemic effectsin patient malignancies. However, leukemia patients often do not haveenough T-cells to collect, meaning that treatment must involve modifiedT cells from donors. Accordingly, there is interest in establishing abank of donor T-cells. Qasim et al. (“First Clinical Application ofTalen Engineered Universal CAR19 T Cells in B-ALL” ASH 57th AnnualMeeting and Exposition, Dec. 5-8, 2015, Abstract 2046(https://ash.confex.com/ash/2015/webprogram/Paper81653.html publishedonline November 2015) discusses modifying CAR19 T cells to eliminate therisk of graft-versus-host disease through the disruption of T-cellreceptor expression and CD52 targeting. Furthermore, CD52 cells weretargeted such that they became insensitive to Alemtuzumab, and thusallowed Alemtuzumab to prevent host-mediated rejection of humanleukocyte antigen (HLA) mismatched CAR19 T-cells. Investigators usedthird generation self-inactivating lentiviral vector encoding a 4 g7CAR19 (CD19 scFv-4-1BB-CD3) linked to RQR8, then electroporated cellswith two pairs of TALEN mRNA for multiplex targeting for both the T-cellreceptor (TCR) alpha constant chain locus and the CD52 gene locus. Cellswhich were still expressing TCR following ex vivo expansion weredepleted using CliniMacs α/β TCR depletion, yielding a T-cell product(UCART19) with <1% TCR expression, 85% of which expressed CAR19, and 64%becoming CD52 negative. The modified CAR19 T cells were administered totreat a patient's relapsed acute lymphoblastic leukemia. The teachingsprovided herein provide effective methods for providing modifiedhematopoietic stem cells and progeny thereof, including but not limitedto cells of the myeloid and lymphoid lineages of blood, including Tcells, B cells, monocytes, macrophages, neutrophils, basophils,eosinophils, erythrocytes, dendritic cells, and megakaryocytes orplatelets, and natural killer cells and their precursors andprogenitors. Such cells can be modified by knocking out, knocking in, orotherwise modulating targets, for example to remove or modulate CD52 asdescribed above, and other targets, such as, without limitation, CXCR4,and PD-1. Thus compositions, cells, and method of the invention can beused to modulate immune responses and to treat, without limitation,malignancies, viral infections, and immune disorders, in conjunctionwith modification of administration of T cells or other cells topatients.

Mention is made of WO 2015/148670 and through the teachings herein theinvention comprehends methods and materials of this document applied inconjunction with the teachings herein. In an aspect of gene therapy,methods and compositions for editing of a target sequence related to orin connection with Human Immunodeficiency Virus (HIV) and AcquiredImmunodeficiency Syndrome (AIDS) are comprehended. In a related aspect,the invention described herein comprehends prevention and treatment ofHIV infection and AIDS, by introducing one or more mutations in the genefor C-C chemokine receptor type 5 (CCR5). The CCR5 gene is also known asCKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22, and CC-CKR-5. In afurther aspect, the invention described herein comprehends provide forprevention or reduction of HIV infection and/or prevention or reductionof the ability for HIV to enter host cells, e.g., in subjects who arealready infected. Exemplary host cells for HIV include, but are notlimited to, CD4 cells, T cells, gut associated lymphatic tissue (GALT),macrophages, dendritic cells, myeloid precursor cell, and microglia.Viral entry into the host cells requires interaction of the viralglycoproteins gp41 and gp120 with both the CD4 receptor and aco-receptor, e.g., CCR5. If a co-receptor, e.g., CCR5, is not present onthe surface of the host cells, the virus cannot bind and enter the hostcells. The progress of the disease is thus impeded. By knocking out orknocking down CCR5 in the host cells, e.g., by introducing a protectivemutation (such as a CCR5 delta 32 mutation), entry of the HIV virus intothe host cells is prevented.

X-linked Chronic granulomatous disease (CGD) is a hereditary disorder ofhost defense due to absent or decreased activity of phagocyte NADPHoxidase. Using a CRISPR-Cas (Cpf1) system that targets and corrects themutation (absent or decreased activity of phagocyte NADPH oxidase)(e.g., with a suitable HDR template that delivers a coding sequence forphagocyte NADPH oxidase); specifically, the gRNA can target mutationthat gives rise to CGD (deficient phagocyte NADPH oxidase), and the HDRcan provide coding for proper expression of phagocyte NADPH oxidase. AngRNA that targets the mutation-and-Cas (Cpf1) protein containingparticle is contacted with HSCs carrying the mutation. The particle alsocan contain a suitable HDR template to correct the mutation for properexpression of phagocyte NADPH oxidase; or the HSC can be contacted witha second particle or a vector that contains or delivers the HDRtemplate. The so contacted cells can be administered; and optionallytreated/expanded; cf. Cartier.

Fanconi anemia: Mutations in at least 15 genes (FANCA, FANCB, FANCC,FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BACH1/BRIP1,FANCL/PHF9/FOG, FANCM, FANCN/PALB2, FANCO/Rad51C, and FANCP/SLX4/BTBD12)can cause Fanconi anemia. Proteins produced from these genes areinvolved in a cell process known as the FA pathway. The FA pathway isturned on (activated) when the process of making new copies of DNA,called DNA replication, is blocked due to DNA damage. The FA pathwaysends certain proteins to the area of damage, which trigger DNA repairso DNA replication can continue. The FA pathway is particularlyresponsive to a certain type of DNA damage known as interstrandcross-links (ICLs). ICLs occur when two DNA building blocks(nucleotides) on opposite strands of DNA are abnormally attached orlinked together, which stops the process of DNA replication. ICLs can becaused by a buildup of toxic substances produced in the body or bytreatment with certain cancer therapy drugs. Eight proteins associatedwith Fanconi anemia group together to form a complex known as the FAcore complex. The FA core complex activates two proteins, called FANCD2and FANCI. The activation of these two proteins brings DNA repairproteins to the area of the ICL so the cross-link can be removed and DNAreplication can continue. the FA core complex. More in particular, theFA core complex is a nuclear multiprotein complex consisting of FANCA,FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, functions as an E3ubiquitin ligase and mediates the activation of the ID complex, which isa heterodimer composed of FANCD2 and FANCI. Once monoubiquitinated, itinteracts with classical tumor suppressors downstream of the FA pathwayincluding FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1, and FANCO/Rad51C andthereby contributes to DNA repair via homologous recombination (HR).Eighty to 90 percent of FA cases are due to mutations in one of threegenes, FANCA, FANCC, and FANCG. These genes provide instructions forproducing components of the FA core complex. Mutations in such genesassociated with the FA core complex will cause the complex to benonfunctional and disrupt the entire FA pathway. As a result, DNA damageis not repaired efficiently and ICLs build up over time. Geiselhart,“Review Article, Disrupted Signaling through the Fanconi Anemia PathwayLeads to Dysfunctional Hematopoietic Stem Cell Biology: UnderlyingMechanisms and Potential Therapeutic Strategies,” Anemia Volume 2012(2012), Article ID 265790, http://dx.doi.org/10.1155/2012/265790discussed FA and an animal experiment involving intrafemoral injectionof a lentivirus encoding the FANCC gene resulting in correction of HSCsin vivo. Using a CRISPR-Cas (Cpf1) system that targets and one or moreof the mutations associated with FA, for instance a CRISPR-Cas (Cpf1)system having gRNA(s) and HDR template(s) that respectively targets oneor more of the mutations of FANCA, FANCC, or FANCG that give rise to FAand provide corrective expression of one or more of FANCA, FANCC orFANCG; e.g., the gRNA can target a mutation as to FANCC, and the HDR canprovide coding for proper expression of FANCC. An gRNA that targets themutation(s) (e.g., one or more involved in FA, such as mutation(s) as toany one or more of FANCA, FANCC or FANCG)-and-Cas (Cpf1) proteincontaining particle is contacted with HSCs carrying the mutation(s). Theparticle also can contain a suitable HDR template(s) to correct themutation for proper expression of one or more of the proteins involvedin FA, such as any one or more of FANCA, FANCC or FANCG; or the HSC canbe contacted with a second particle or a vector that contains ordelivers the HDR template. The so contacted cells can be administered;and optionally treated/expanded; cf. Cartier.

The particle in the herein discussion (e.g., as to containing gRNA(s)and Cas (Cpf1), optionally HDR template(s), or HDR template(s); forinstance as to Hemophilia B, SCID, SCID-X1, ADA-SCID, Hereditarytyrosinemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome,Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy(MLD), HIV/AIDS, Immunodeficiency disorder, Hematologic condition, orgenetic lysosomal storage disease) is advantageously obtained orobtainable from admixing an gRNA(s) and Cas (Cpf1) protein mixture(optionally containing HDR template(s) or such mixture only containingHDR template(s) when separate particles as to template(s) is desired)with a mixture comprising or consisting essentially of or consisting ofsurfactant, phospholipid, biodegradable polymer, lipoprotein and alcohol(wherein one or more gRNA targets the genetic locus or loci in the HSC).

Indeed, the invention is especially suited for treating hematopoieticgenetic disorders with genome editing, and immunodeficiency disorders,such as genetic immunodeficiency disorders, especially through using theparticle technology herein-discussed. Genetic immunodeficiencies arediseases where genome editing interventions of the instant invention cansuccessful. The reasons include: Hematopoietic cells, of which immunecells are a subset, are therapeutically accessible. They can be removedfrom the body and transplanted autologously or allogenically. Further,certain genetic immunodeficiencies, e.g., severe combinedimmunodeficiency (SCID), create a proliferative disadvantage for immunecells. Correction of genetic lesions causing SCID by rare, spontaneous‘reverse’ mutations indicates that correcting even one lymphocyteprogenitor may be sufficient to recover immune function inpatients.../../../Users/t_kowalski/AppData/Local/Microsoft/Windows/TemporaryInternet Files/Content.Outlook/GA8VY8LK/Treating SCID forEllen.docx-_ENREF_1 See Bousso, P., et al. Diversity, functionality, andstability of the T cell repertoire derived in vivo from a single human Tcell precursor. Proceedings of the National Academy of Sciences of theUnited States of America 97, 274-278 (2000). The selective advantage foredited cells allows for even low levels of editing to result in atherapeutic effect. This effect of the instant invention can be seen inSCID, Wiskott-Aldrich Syndrome, and the other conditions mentionedherein, including other genetic hematopoietic disorders such as alpha-and beta-thalassemia, where hemoglobin deficiencies negatively affectthe fitness of erythroid progenitors.

The activity of NHEJ and HDR DSB repair varies significantly by celltype and cell state. NHEJ is not highly regulated by the cell cycle andis efficient across cell types, allowing for high levels of genedisruption in accessible target cell populations. In contrast, HDR actsprimarily during S/G2 phase, and is therefore restricted to cells thatare actively dividing, limiting treatments that require precise genomemodifications to mitotic cells [Ciccia, A. & Elledge, S. J. Molecularcell 40, 179-204 (2010); Chapman, J. R., et al. Molecular cell 47,497-510 (2012)].

The efficiency of correction via HDR may be controlled by the epigeneticstate or sequence of the targeted locus, or the specific repair templateconfiguration (single vs. double stranded, long vs. short homology arms)used [Hacein-Bey-Abina, S., et al. The New England journal of medicine346, 1185-1193 (2002); Gaspar, H. B., et al. Lancet 364, 2181-2187(2004); Beumer, K. J., et al. G3 (2013)]. The relative activity of NHEJand HDR machineries in target cells may also affect gene correctionefficiency, as these pathways may compete to resolve DSBs [Beumer, K.J., et al. Proceedings of the National Academy of Sciences of the UnitedStates of America 105, 19821-19826 (2008)]. HDR also imposes a deliverychallenge not seen with NHEJ strategies, as it requires the concurrentdelivery of nucleases and repair templates. In practice, theseconstraints have so far led to low levels of HDR in therapeuticallyrelevant cell types. Clinical translation has therefore largely focusedon NHEJ strategies to treat disease, although proof-of-conceptpreclinical HDR treatments have now been described for mouse models ofhaemophilia B and hereditary tyrosinemia [Li, H., et al. Nature 475,217-221 (2011); Yin, H., et al. Nature biotechnology 32, 551-553(2014)].

Any given genome editing application may comprise combinations ofproteins, small RNA molecules, and/or repair templates, making deliveryof these multiple parts substantially more challenging than smallmolecule therapeutics. Two main strategies for delivery of genomeediting tools have been developed: ex vivo and in vivo. In ex vivotreatments, diseased cells are removed from the body, edited and thentransplanted back into the patient. Ex vivo editing has the advantage ofallowing the target cell population to be well defined and the specificdosage of therapeutic molecules delivered to cells to be specified. Thelatter consideration may be particularly important when off-targetmodifications are a concern, as titrating the amount of nuclease maydecrease such mutations (Hsu et al., 2013). Another advantage of ex vivoapproaches is the typically high editing rates that can be achieved, dueto the development of efficient delivery systems for proteins andnucleic acids into cells in culture for research and gene therapyapplications.

There may be drawbacks with ex vivo approaches that limit application toa small number of diseases. For instance, target cells must be capableof surviving manipulation outside the body. For many tissues, like thebrain, culturing cells outside the body is a major challenge, becausecells either fail to survive, or lose properties necessary for theirfunction in vivo. Thus, in view of this disclosure and the knowledge inthe art, ex vivo therapy as to tissues with adult stem cell populationsamenable to ex vivo culture and manipulation, such as the hematopoieticsystem, by the CRISPR-Cas (Cpf1) system are enabled. [Bunn, H. F. &Aster, J. Pathophysiology of blood disorders, (McGraw-Hill, New York,2011)]

In vivo genome editing involves direct delivery of editing systems tocell types in their native tissues. In vivo editing allows diseases inwhich the affected cell population is not amenable to ex vivomanipulation to be treated. Furthermore, delivering nucleases to cellsin situ allows for the treatment of multiple tissue and cell types.These properties probably allow in vivo treatment to be applied to awider range of diseases than ex vivo therapies.

To date, in vivo editing has largely been achieved through the use ofviral vectors with defined, tissue-specific tropism. Such vectors arecurrently limited in terms of cargo carrying capacity and tropism,restricting this mode of therapy to organ systems where transductionwith clinically useful vectors is efficient, such as the liver, muscleand eye [Kotterman, M. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Nguyen, T. H. & Ferry, N. Gene therapy 11 Suppl 1,S76-84 (2004); Boye, S. E., et al. Molecular therapy: the journal of theAmerican Society of Gene Therapy 21, 509-519 (2013)].

A potential barrier for in vivo delivery is the immune response that maybe created in response to the large amounts of virus necessary fortreatment, but this phenomenon is not unique to genome editing and isobserved with other virus based gene therapies [Bessis, N., et al. Genetherapy 11 Suppl 1, S10-17 (2004)]. It is also possible that peptidesfrom editing nucleases themselves are presented on WIC Class I moleculesto stimulate an immune response, although there is little evidence tosupport this happening at the preclinical level. Another majordifficulty with this mode of therapy is controlling the distribution andconsequently the dosage of genome editing nucleases in vivo, leading tooff-target mutation profiles that may be difficult to predict. However,in view of this disclosure and the knowledge in the art, including theuse of virus- and particle-based therapies being used in the treatmentof cancers, in vivo modification of HSCs, for instance by delivery byeither particle or virus, is within the ambit of the skilled person.

Ex Vivo Editing Therapy: The long standing clinical expertise with thepurification, culture and transplantation of hematopoietic cells hasmade diseases affecting the blood system such as SCID, Fanconi anemia,Wiskott-Aldrich syndrome and sickle cell anemia the focus of ex vivoediting therapy. Another reason to focus on hematopoietic cells is that,thanks to previous efforts to design gene therapy for blood disorders,delivery systems of relatively high efficiency already exist. With theseadvantages, this mode of therapy can be applied to diseases where editedcells possess a fitness advantage, so that a small number of engrafted,edited cells can expand and treat disease. One such disease is HIV,where infection results in a fitness disadvantage to CD4+ T cells.

Ex vivo editing therapy has been recently extended to include genecorrection strategies. The barriers to HDR ex vivo were overcome in arecent paper from Genovese and colleagues, who achieved gene correctionof a mutated IL2RG gene in hematopoietic stem cells (HSCs) obtained froma patient suffering from SCID-X1 [Genovese, P., et al. Nature 510,235-240 (2014)]. Genovese et. al. accomplished gene correction in HSCsusing a multimodal strategy. First, HSCs were transduced usingintegration-deficient lentivirus containing an HDR template encoding atherapeutic cDNA for IL2RG. Following transduction, cells wereelectroporated with mRNA encoding ZFNs targeting a mutational hotspot inIL2RG to stimulate HDR based gene correction. To increase HDR rates,culture conditions were optimized with small molecules to encourage HSCdivision. With optimized culture conditions, nucleases and HDRtemplates, gene corrected HSCs from the SCID-X1 patient were obtained inculture at therapeutically relevant rates. HSCs from unaffectedindividuals that underwent the same gene correction procedure couldsustain long-term hematopoiesis in mice, the gold standard for HSCfunction. HSCs are capable of giving rise to all hematopoietic celltypes and can be autologously transplanted, making them an extremelyvaluable cell population for all hematopoietic genetic disorders[Weissman, I. L. & Shizuru, J. A. Blood 112, 3543-3553 (2008)]. Genecorrected HSCs could, in principle, be used to treat a wide range ofgenetic blood disorders making this study an exciting breakthrough fortherapeutic genome editing.

In Vivo Editing Therapy: In vivo editing can be used advantageously fromthis disclosure and the knowledge in the art. For organ systems wheredelivery is efficient, there have already been a number of excitingpreclinical therapeutic successes. The first example of successful invivo editing therapy was demonstrated in a mouse model of haemophilia B[Li, H., et al. Nature 475, 217-221 (2011)]. As noted earlier,Haemophilia B is an X-linked recessive disorder caused byloss-of-function mutations in the gene encoding Factor IX, a crucialcomponent of the clotting cascade. Recovering Factor IX activity toabove 1% of its levels in severely affected individuals can transformthe disease into a significantly milder form, as infusion of recombinantFactor IX into such patients prophylactically from a young age toachieve such levels largely ameliorates clinical complications[Lofqvist, T., et al. Journal of internal medicine 241, 395-400 (1997)].Thus, only low levels of HDR gene correction are necessary to changeclinical outcomes for patients. In addition, Factor IX is synthesizedand secreted by the liver, an organ that can be transduced efficientlyby viral vectors encoding editing systems.

Using hepatotropic adeno-associated viral (AAV) serotypes encoding ZFNsand a corrective HDR template, up to 7% gene correction of a mutated,humanized Factor IX gene in the murine liver was achieved [Li, H., etal. Nature 475, 217-221 (2011)]. This resulted in improvement of clotformation kinetics, a measure of the function of the clotting cascade,demonstrating for the first time that in vivo editing therapy is notonly feasible, but also efficacious. As discussed herein, the skilledperson is positioned from the teachings herein and the knowledge in theart, e.g., Li to address Haemophilia B with a particle-containing HDRtemplate and a CRISPR-Cas (Cpf1) system that targets the mutation of theX-linked recessive disorder to reverse the loss-of-function mutation.

Building on this study, other groups have recently used in vivo genomeediting of the liver with CRISPR-Cas to successfully treat a mouse modelof hereditary tyrosinemia and to create mutations that provideprotection against cardiovascular disease. These two distinctapplications demonstrate the versatility of this approach for disordersthat involve hepatic dysfunction [Yin, H., et al. Nature biotechnology32, 551-553 (2014); Ding, Q., et al. Circulation research 115, 488-492(2014)]. Application of in vivo editing to other organ systems arenecessary to prove that this strategy is widely applicable. Currently,efforts to optimize both viral and non-viral vectors are underway toexpand the range of disorders that can be treated with this mode oftherapy [Kotterman, M. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Yin, H., et al. Nature reviews. Genetics 15, 541-555(2014)]. As discussed herein, the skilled person is positioned from theteachings herein and the knowledge in the art, e.g., Yin to addresshereditary tyrosinemia with a particle-containing HDR template and aCRISPR-Cas (Cpf1) system that targets the mutation.

Targeted deletion, therapeutic applications: Targeted deletion of genesmay be preferred. Preferred are, therefore, genes involved inimmunodeficiency disorder, hematologic condition, or genetic lysosomalstorage disease, e.g., Hemophilia B, SCID, SCID-X1, ADA-SCID, Hereditarytyrosinemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome,Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy(MLD), HIV/AIDS, other metabolic disorders, genes encoding mis-foldedproteins involved in diseases, genes leading to loss-of-functioninvolved in diseases; generally, mutations that can be targeted in anHSC, using any herein-discussed delivery system, with the particlesystem considered advantageous.

In the present invention, the immunogenicity of the CRISPR enzyme inparticular may be reduced following the approach first set out in Tangriet al with respect to erythropoietin and subsequently developed.Accordingly, directed evolution or rational design may be used to reducethe immunogenicity of the CRISPR enzyme (for instance a Cpf1) in thehost species (human or other species).

Genome editing: The CRISPR/Cas (Cpf1) systems of the present inventioncan be used to correct genetic mutations that were previously attemptedwith limited success using TALEN and ZFN and lentiviruses, including asherein discussed; see also WO2013163628.

Treating Disease of the Brain, Central Nervous and Immune Systems

The present invention also contemplates delivering the CRISPR-Cas systemto the brain or neurons. For example, RNA interference (RNAi) offerstherapeutic potential for this disorder by reducing the expression ofHTT, the disease-causing gene of Huntington's disease (see, e.g.,McBride et al., Molecular Therapy vol. 19 no. 12 Dec. 2011, pp.2152-2162), therefore Applicant postulates that it may be used/and oradapted to the CRISPR-Cas system. The CRISPR-Cas system may be generatedusing an algorithm to reduce the off-targeting potential of antisensesequences. The CRISPR-Cas sequences may target either a sequence in exon52 of mouse, rhesus or human huntingtin and expressed in a viral vector,such as AAV. Animals, including humans, may be injected with about threemicroinjections per hemisphere (six injections total): the first 1 mmrostral to the anterior commissure (12 μl) and the two remaininginjections (12 μl and 10 μl, respectively) spaced 3 and 6 mm caudal tothe first injection with 1e12 vg/ml of AAV at a rate of about 1μl/minute, and the needle was left in place for an additional 5 minutesto allow the injectate to diffuse from the needle tip.

DiFiglia et al. (PNAS, Oct. 23, 2007, vol. 104, no. 43, 17204-17209)observed that single administration into the adult striatum of an siRNAtargeting Htt can silence mutant Htt, attenuate neuronal pathology, anddelay the abnormal behavioral phenotype observed in a rapid-onset, viraltransgenic mouse model of HD. DiFiglia injected mice intrastriatallywith 2 μl of Cy3-labeled cc-siRNA-Htt or unconjugated siRNA-Htt at 10μM. A similar dosage of CRISPR Cas targeted to Htt may be contemplatedfor humans in the present invention, for example, about 5-10 ml of 10 μMCRISPR Cas targeted to Htt may be injected intrastriatally.

In another example, Boudreau et al. (Molecular Therapy vol. 17 no. 6Jun. 2009) injects 5 μl of recombinant AAV serotype 2/1 vectorsexpressing htt-specific RNAi virus (at 4×10¹² viral genomes/ml) into thestraiatum. A similar dosage of CRISPR Cas targeted to Htt may becontemplated for humans in the present invention, for example, about10-20 ml of 4×10¹² viral genomes/ml) CRISPR Cas targeted to Htt may beinjected intrastriatally.

In another example, a CRISPR Cas targeted to HTT may be administeredcontinuously (see, e.g., Yu et al., Cell 150, 895-908, Aug. 31, 2012).Yu et al. utilizes osmotic pumps delivering 0.25 ml/hr (Model 2004) todeliver 300 mg/day of ss-siRNA or phosphate-buffered saline (PBS) (SigmaAldrich) for 28 days, and pumps designed to deliver 0.5 μl/hr (Model2002) were used to deliver 75 mg/day of the positive control MOE ASO for14 days. Pumps (Durect Corporation) were filled with ss-siRNA or MOEdiluted in sterile PBS and then incubated at 37 C for 24 or 48 (Model2004) hours prior to implantation. Mice were anesthetized with 2.5%isofluorane, and a midline incision was made at the base of the skull.Using stereotaxic guides, a cannula was implanted into the right lateralventricle and secured with Loctite adhesive. A catheter attached to anAlzet osmotic mini pump was attached to the cannula, and the pump wasplaced subcutaneously in the midscapular area. The incision was closedwith 5.0 nylon sutures. A similar dosage of CRISPR Cas targeted to Httmay be contemplated for humans in the present invention, for example,about 500 to 1000 g/day CRISPR Cas targeted to Htt may be administered.

In another example of continuous infusion, Stiles et al. (ExperimentalNeurology 233 (2012) 463-471) implanted an intraparenchymal catheterwith a titanium needle tip into the right putamen. The catheter wasconnected to a SynchroMed® II Pump (Medtronic Neurological, Minneapolis,Minn.) subcutaneously implanted in the abdomen. After a 7 day infusionof phosphate buffered saline at 6 μL/day, pumps were re-filled with testarticle and programmed for continuous delivery for 7 days. About 2.3 to11.52 mg/d of siRNA were infused at varying infusion rates of about 0.1to 0.5 μL/min. A similar dosage of CRISPR Cas targeted to Htt may becontemplated for humans in the present invention, for example, about 20to 200 mg/day CRISPR Cas targeted to Htt may be administered. In anotherexample, the methods of US Patent Publication No. 20130253040 assignedto Sangamo may also be also be adapted from TALES to the nucleicacid-targeting system of the present invention for treating Huntington'sDisease.

In another example, the methods of US Patent Publication No. 20130253040(WO2013130824) assigned to Sangamo may also be also be adapted fromTALES to the CRISPR Cas system of the present invention for treatingHuntington's Disease.

WO2015089354 A1 in the name of The Broad Institute et al., herebyincorporated by reference, describes a targets for Huntington's Disease(HP). Possible target genes of CRISPR complex in regard to Huntington'sDisease: PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; and TGM2. Accordingly,one or more of PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; and TGM2 may beselected as targets for Huntington's Disease in some embodiments of thepresent invention.

Other trinucleotide repeat disorders. These may include any of thefollowing: Category I includes Huntington's disease (HD) and thespinocerebellar ataxias; Category II expansions are phenotypicallydiverse with heterogeneous expansions that are generally small inmagnitude, but also found in the exons of genes; and Category IIIincludes fragile X syndrome, myotonic dystrophy, two of thespinocerebellar ataxias, juvenile myoclonic epilepsy, and Friedreich'sataxia.

A further aspect of the invention relates to utilizing the CRISPR-Cassystem for correcting defects in the EMP2A and EMP2B genes that havebeen identified to be associated with Lafora disease. Lafora disease isan autosomal recessive condition which is characterized by progressivemyoclonus epilepsy which may start as epileptic seizures in adolescence.A few cases of the disease may be caused by mutations in genes yet to beidentified. The disease causes seizures, muscle spasms, difficultywalking, dementia, and eventually death. There is currently no therapythat has proven effective against disease progression. Other geneticabnormalities associated with epilepsy may also be targeted by theCRISPR-Cas system and the underlying genetics is further described inGenetics of Epilepsy and Genetic Epilepsies, edited by GiulianoAvanzini, Jeffrey L. Noebels, Mariani Foundation PaediatricNeurology:20; 2009).

The methods of US Patent Publication No. 20110158957 assigned to SangamoBioSciences, Inc. involved in inactivating T cell receptor (TCR) genesmay also be modified to the CRISPR Cas system of the present invention.In another example, the methods of US Patent Publication No. 20100311124assigned to Sangamo BioSciences, Inc. and US Patent Publication No.20110225664 assigned to Cellectis, which are both involved ininactivating glutamine synthetase gene expression genes may also bemodified to the CRISPR Cas system of the present invention.

Delivery options for the brain include encapsulation of CRISPR enzymeand guide RNA in the form of either DNA or RNA into liposomes andconjugating to molecular Trojan horses for trans-blood brain barrier(BBB) delivery. Molecular Trojan horses have been shown to be effectivefor delivery of B-gal expression vectors into the brain of non-humanprimates. The same approach can be used to delivery vectors containingCRISPR enzyme and guide RNA. For instance, Xia C F and Boado R J,Pardridge W M (“Antibody-mediated targeting of siRNA via the humaninsulin receptor using avidin-biotin technology.” Mol Pharm. 2009May-June; 6(3):747-51. doi: 10.1021/mp800194) describes how delivery ofshort interfering RNA (siRNA) to cells in culture, and in vivo, ispossible with combined use of a receptor-specific monoclonal antibody(mAb) and avidin-biotin technology. The authors also report that becausethe bond between the targeting mAb and the siRNA is stable withavidin-biotin technology, and RNAi effects at distant sites such asbrain are observed in vivo following an intravenous administration ofthe targeted siRNA.

Zhang et al. (Mol Ther. 2003 January; 7(1):11-8)) describe howexpression plasmids encoding reporters such as luciferase wereencapsulated in the interior of an “artificial virus” comprised of an 85nm pegylated immunoliposome, which was targeted to the rhesus monkeybrain in vivo with a monoclonal antibody (MAb) to the human insulinreceptor (HIR). The HIRMAb enables the liposome carrying the exogenousgene to undergo transcytosis across the blood-brain barrier andendocytosis across the neuronal plasma membrane following intravenousinjection. The level of luciferase gene expression in the brain was50-fold higher in the rhesus monkey as compared to the rat. Widespreadneuronal expression of the beta-galactosidase gene in primate brain wasdemonstrated by both histochemistry and confocal microscopy. The authorsindicate that this approach makes feasible reversible adult transgenicsin 24 hours. Accordingly, the use of immunoliposome is preferred. Thesemay be used in conjunction with antibodies to target specific tissues orcell surface proteins.

Alzheimer's Disease

US Patent Publication No. 20110023153, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith Alzheimer's Disease. Once modified cells and animals may be furthertested using known methods to study the effects of the targetedmutations on the development and/or progression of AD using measurescommonly used in the study of AD—such as, without limitation, learningand memory, anxiety, depression, addiction, and sensory motor functionsas well as assays that measure behavioral, functional, pathological,metaboloic and biochemical function.

The present disclosure comprises editing of any chromosomal sequencesthat encode proteins associated with AD. The AD-related proteins aretypically selected based on an experimental association of theAD-related protein to an AD disorder. For example, the production rateor circulating concentration of an AD-related protein may be elevated ordepressed in a population having an AD disorder relative to a populationlacking the AD disorder. Differences in protein levels may be assessedusing proteomic techniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the AD-related proteins may beidentified by obtaining gene expression profiles of the genes encodingthe proteins using genomic techniques including but not limited to DNAmicroarray analysis, serial analysis of gene expression (SAGE), andquantitative real-time polymerase chain reaction (Q-PCR).

Examples of Alzheimer's disease associated proteins may include the verylow density lipoprotein receptor protein (VLDLR) encoded by the VLDLRgene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded bythe UBA1 gene, or the NEDD8-activating enzyme E1 catalytic subunitprotein (UBE1C) encoded by the UBA3 gene, for example.

By way of non-limiting example, proteins associated with AD include butare not limited to the proteins listed as follows: Chromosomal SequenceEncoded Protein ALAS2 Delta-aminolevulinate synthase 2 (ALAS2) ABCA1ATP-binding cassette transporter (ABCA1) ACE Angiotensin I-convertingenzyme (ACE) APOE Apolipoprotein E precursor (APOE) APP amyloidprecursor protein (APP) AQP1 aquaporin 1 protein (AQP1) BIN1 Mycbox-dependent-interacting protein 1 or bridging integrator 1 protein(BIN1) BDNF brain-derived neurotrophic factor (BDNF) BTNL8Butyrophilin-like protein 8 (BTNL8) C1ORF49 chromosome 1 open readingframe 49 CDH4 Cadherin-4 CHRNB2 Neuronal acetylcholine receptor subunitbeta-2 CKLFSF2 CKLF-like MARVEL transmembrane domain-containing protein2 (CKLFSF2) CLEC4E C-type lectin domain family 4, member e (CLEC4E) CLUclusterin protein (also known as apoplipoprotein J) CR1 Erythrocytecomplement receptor 1 (CR1, also known as CD35, C3b/C4b receptor andimmune adherence receptor) CR1L Erythrocyte complement receptor 1 (CR1L)CSF3R granulocyte colony-stimulating factor 3 receptor (CSF3R) CST3Cystatin C or cystatin 3 CYP2C Cytochrome P450 2C DAPK1 Death-associatedprotein kinase 1 (DAPK1) ESR1 Estrogen receptor 1 FCAR Fc fragment ofIgA receptor (FCAR, also known as CD89) FCGR3B Fc fragment of IgG, lowaffinity Mb, receptor (FCGR3B or CD16b) FFA2 Free fatty acid receptor 2(FFA2) FGA Fibrinogen (Factor I) GAB2 GRB2-associated-binding protein 2(GAB2) GAB2 GRB2-associated-binding protein 2 (GAB2) GALP Galanin-likepeptide GAPDHS Glyceraldehyde-3-phosphate dehydrogenase, spermatogenic(GAPDHS) GMPB GMBP HP Haptoglobin (HP) HTR7 5-hydroxytryptamine(serotonin) receptor 7 (adenylate cyclase-coupled) IDE Insulin degradingenzyme IF127 IF127 IFI6 Interferon, alpha-inducible protein 6 (IFI6)IFIT2 Interferon-induced protein with tetratricopeptide repeats 2(IFIT2) IL1RN interleukin-1 receptor antagonist (IL-1RA) IL8RAInterleukin 8 receptor, alpha (IL8RA or CD181) IL8RB Interleukin 8receptor, beta (IL8RB) JAG1 Jagged 1 (JAG1) KCNJ15 Potassiuminwardly-rectifying channel, subfamily J, member 15 (KCNJ15) LRP6Low-density lipoprotein receptor-related protein 6 (LRP6) MAPTmicrotubule-associated protein tau (MAPT) MARK4 MAP/microtubuleaffinity-regulating kinase 4 (MARK4) MPHOSPH1 M-phase phosphoprotein 1MTHFR 5,10-methylenetetrahydrofolate reductase MX2 Interferon-inducedGTP-binding protein Mx2 NBN Nibrin, also known as NBN NCSTN NicastrinNIACR2 Niacin receptor 2 (NIACR2, also known as GPR109B) NMNAT3nicotinamide nucleotide adenylyltransferase 3 NTM Neurotrimin (or HNT)ORM1 Orosmucoid 1 (ORM1) or Alpha-1-acid glycoprotein 1 P2RY13 P2Ypurinoceptor 13 (P2RY13) PBEF1 Nicotinamide phosphoribosyltransferase(NAmPRTase or Nampt) also known as pre-B-cell colony-enhancing factor 1(PBEF1) or visfatin PCK1 Phosphoenolpyruvate carboxykinase PICALMphosphatidylinositol binding clathrin assembly protein (PICALM) PLAUUrokinase-type plasminogen activator (PLAU) PLXNC1 Plexin C1 (PLXNC1)PRNP Prion protein PSEN1 presenilin 1 protein (PSEN1) PSEN2 presenilin 2protein (PSEN2) PTPRA protein tyrosine phosphatase receptor type Aprotein (PTPRA) RALGPS2 Ral GEF with PH domain and SH3 binding motif 2(RALGPS2) RGSL2 regulator of G-protein signaling like 2 (RGSL2) SELENBP1Selenium binding protein 1 (SELNBP1) SLC25A37 Mitoferrin-1 SORL1sortilin-related receptor L(DLR class) A repeats-containing protein(SORL1) TF Transferrin TFAM Mitochondrial transcription factor A TNFTumor necrosis factor TNFRSF10C Tumor necrosis factor receptorsuperfamily member 10C (TNFRSF10C) TNFSF10 Tumor necrosis factorreceptor superfamily, (TRAIL) member 10a (TNFSF10) UBA1 ubiquitin-likemodifier activating enzyme 1 (UBA1) UBA3 NEDD8-activating enzyme E1catalytic subunit protein (UBE1C) UBB ubiquitin B protein (UBB) UBQLN1Ubiquilin-1 UCHL1 ubiquitin carboxyl-terminal esterase L1 protein(UCHL1) UCHL3 ubiquitin carboxyl-terminal hydrolase isozyme L3 protein(UCHL3) VLDLR very low density lipoprotein receptor protein (VLDLR)

In exemplary embodiments, the proteins associated with AD whosechromosomal sequence is edited may be the very low density lipoproteinreceptor protein (VLDLR) encoded by the VLDLR gene, the ubiquitin-likemodifier activating enzyme 1 (UBA1) encoded by the UBA1 gene, theNEDD8-activating enzyme E1 catalytic subunit protein (UBE1C) encoded bythe UBA3 gene, the aquaporin 1 protein (AQP1) encoded by the AQP1 gene,the ubiquitin carboxyl-terminal esterase L1 protein (UCHL1) encoded bythe UCHL1 gene, the ubiquitin carboxyl-terminal hydrolase isozyme L3protein (UCHL3) encoded by the UCHL3 gene, the ubiquitin B protein (UBB)encoded by the UBB gene, the microtubule-associated protein tau (MAPT)encoded by the MAPT gene, the protein tyrosine phosphatase receptor typeA protein (PTPRA) encoded by the PTPRA gene, the phosphatidylinositolbinding clathrin assembly protein (PICALM) encoded by the PICALM gene,the clusterin protein (also known as apoplipoprotein J) encoded by theCLU gene, the presenilin 1 protein encoded by the PSEN1 gene, thepresenilin 2 protein encoded by the PSEN2 gene, the sortilin-relatedreceptor L(DLR class) A repeats-containing protein (SORL1) proteinencoded by the SORL1 gene, the amyloid precursor protein (APP) encodedby the APP gene, the Apolipoprotein E precursor (APOE) encoded by theAPOE gene, or the brain-derived neurotrophic factor (BDNF) encoded bythe BDNF gene. In an exemplary embodiment, the genetically modifiedanimal is a rat, and the edited chromosomal sequence encoding theprotein associated with AD is as follows: APP amyloid precursor protein(APP) NM_019288 AQP1 aquaporin 1 protein (AQP1) NM_012778 BDNFBrain-derived neurotrophic factor NM_012513 CLU clusterin protein (alsoknown as NM_053021 apoplipoprotein J) MAPT microtubule-associatedprotein NM_017212 tau (MAPT) PICALM phosphatidylinositol bindingNM_053554 clathrin assembly protein (PICALM) PSEN1 presenilin 1 protein(PSEN1) NM_019163 PSEN2 presenilin 2 protein (PSEN2) NM_031087 PTPRAprotein tyrosine phosphatase NM_012763 receptor type A protein (PTPRA)SORL1 sortilin-related receptor L(DLR NM_053519, class) Arepeats-containing XM_001065506, protein (SORL1) XM_217115 UBA1ubiquitin-like modifier activating NM_001014080 enzyme 1 (UBA1) UBA3NEDD8-activating enzyme E1 NM_057205 catalytic subunit protein (UBE1C)UBB ubiquitin B protein (UBB) NM_138895 UCHL1 ubiquitincarboxyl-terminal NM_017237 esterase L1 protein (UCHL1) UCHL3 ubiquitincarboxyl-terminal NM_001110165 hydrolase isozyme L3 protein (UCHL3)VLDLR very low density lipoprotein NM_013155 receptor protein (VLDLR)

The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or more disrupted chromosomal sequences encoding a proteinassociated with AD and zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or more chromosomally integrated sequences encoding a proteinassociated with AD.

The edited or integrated chromosomal sequence may be modified to encodean altered protein associated with AD. A number of mutations inAD-related chromosomal sequences have been associated with AD. Forinstance, the V7171 (i.e. valine at position 717 is changed toisoleucine) missense mutation in APP causes familial AD. Multiplemutations in the presenilin-1 protein, such as H163R (i.e. histidine atposition 163 is changed to arginine), A246E (i.e. alanine at position246 is changed to glutamate), L286V (i.e. leucine at position 286 ischanged to valine) and C410Y (i.e. cysteine at position 410 is changedto tyrosine) cause familial Alzheimer's type 3. Mutations in thepresenilin-2 protein, such as N141 I (i.e. asparagine at position 141 ischanged to isoleucine), M239V (i.e. methionine at position 239 ischanged to valine), and D439A (i.e. aspartate at position 439 is changedto alanine) cause familial Alzheimer's type 4. Other associations ofgenetic variants in AD-associated genes and disease are known in theart. See, for example, Waring et al. (2008) Arch. Neurol. 65:329-334,the disclosure of which is incorporated by reference herein in itsentirety.

Secretase Disorders

US Patent Publication No. 20110023146, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith secretase-associated disorders. Secretases are essential forprocessing pre-proteins into their biologically active forms. Defects invarious components of the secretase pathways contribute to manydisorders, particularly those with hallmark amyloidogenesis or amyloidplaques, such as Alzheimer's disease (AD).

A secretase disorder and the proteins associated with these disordersare a diverse set of proteins that effect susceptibility for numerousdisorders, the presence of the disorder, the severity of the disorder,or any combination thereof. The present disclosure comprises editing ofany chromosomal sequences that encode proteins associated with asecretase disorder. The proteins associated with a secretase disorderare typically selected based on an experimental association of thesecretase-related proteins with the development of a secretase disorder.For example, the production rate or circulating concentration of aprotein associated with a secretase disorder may be elevated ordepressed in a population with a secretase disorder relative to apopulation without a secretase disorder. Differences in protein levelsmay be assessed using proteomic techniques including but not limited toWestern blot, immunohistochemical staining, enzyme linked immunosorbentassay (ELISA), and mass spectrometry. Alternatively, the proteinassociated with a secretase disorder may be identified by obtaining geneexpression profiles of the genes encoding the proteins using genomictechniques including but not limited to DNA microarray analysis, serialanalysis of gene expression (SAGE), and quantitative real-timepolymerase chain reaction (Q-PCR).

By way of non-limiting example, proteins associated with a secretasedisorder include PSENEN (presenilin enhancer 2 homolog (C. elegans)),CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4)precursor protein), APH1B (anterior pharynx defective 1 homolog B (C.elegans)), PSEN2 (presenilin 2 (Alzheimer disease 4)), BACE1 (beta-siteAPP-cleaving enzyme 1), ITM2B (integral membrane protein 2B), CTSD(cathepsin D), NOTCH1 (Notch homolog 1, translocation-associated(Drosophila)), TNF (tumor necrosis factor (TNF superfamily, member 2)),INS (insulin), DYT10 (dystonia 10), ADAM17 (ADAM metallopeptidase domain17), APOE (apolipoprotein E), ACE (angiotensin I converting enzyme(peptidyl-dipeptidase A) 1), STN (statin), TP53 (tumor protein p53), IL6(interleukin 6 (interferon, beta 2)), NGFR (nerve growth factor receptor(TNFR superfamily, member 16)), IL1B (interleukin 1, beta), ACHE(acetylcholinesterase (Yt blood group)), CTNNB1 (catenin(cadherin-associated protein), beta 1, 88 kDa), IGF1 (insulin-likegrowth factor 1 (somatomedin C)), IFNG (interferon, gamma), NRG1(neuregulin 1), CASP3 (caspase 3, apoptosis-related cysteine peptidase),MAPK1 (mitogen-activated protein kinase 1), CDH1 (cadherin 1, type 1,E-cadherin (epithelial)), APBB1 (amyloid beta (A4) precursorprotein-binding, family B, member 1 (Fe65)), HMGCR(3-hydroxy-3-methylglutaryl-Coenzyme A reductase), CREB1 (cAMPresponsive element binding protein 1), PTGS2 (prostaglandin-endoperoxidesynthase 2 (prostaglandin G/H synthase and cyclooxygenase)), HES1 (hairyand enhancer of split 1, (Drosophila)), CAT (catalase), TGFB1(transforming growth factor, beta 1), ENO2 (enolase 2 (gamma,neuronal)), ERBB4 (v-erb-a erythroblastic leukemia viral oncogenehomolog 4 (avian)), TRAPPC10 (trafficking protein particle complex 10),MAOB (monoamine oxidase B), NGF (nerve growth factor (betapolypeptide)), MMP12 (matrix metallopeptidase 12 (macrophage elastase)),JAG1 (jagged 1 (Alagille syndrome)), CD40LG (CD40 ligand), PPARG(peroxisome proliferator-activated receptor gamma), FGF2 (fibroblastgrowth factor 2 (basic)), IL3 (interleukin 3 (colony-stimulating factor,multiple)), LRP1 (low density lipoprotein receptor-related protein 1),NOTCH4 (Notch homolog 4 (Drosophila)), MAPK8 (mitogen-activated proteinkinase 8), PREP (prolyl endopeptidase), NOTCH3 (Notch homolog 3(Drosophila)), PRNP (prion protein), CTSG (cathepsin G), EGF (epidermalgrowth factor (beta-urogastrone)), REN (renin), CD44 (CD44 molecule(Indian blood group)), SELP (selectin P (granule membrane protein 140kDa, antigen CD62)), GHR (growth hormone receptor), ADCYAP1 (adenylatecyclase activating polypeptide 1 (pituitary)), INSR (insulin receptor),GFAP (glial fibrillary acidic protein), MMP3 (matrix metallopeptidase 3(stromelysin 1, progelatinase)), MAPK10 (mitogen-activated proteinkinase 10), SP1 (Sp1 transcription factor), MYC (v-myc myelocytomatosisviral oncogene homolog (avian)), CTSE (cathepsin E), PPARA (peroxisomeproliferator-activated receptor alpha), JUN (jun oncogene), TIMP1 (TIMPmetallopeptidase inhibitor 1), IL5 (interleukin 5 (colony-stimulatingfactor, eosinophil)), ILIA (interleukin 1, alpha), MMP9 (matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase)), HTR4 (5-hydroxytryptamine (serotonin) receptor 4), HSPG2(heparan sulfate proteoglycan 2), KRAS (v-Ki-ras2 Kirsten rat sarcomaviral oncogene homolog), CYCS (cytochrome c, somatic), SMG1 (SMG1homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans)),IL1R1 (interleukin 1 receptor, type I), PROK1 (prokineticin 1), MAPK3(mitogen-activated protein kinase 3), NTRK1 (neurotrophic tyrosinekinase, receptor, type 1), IL13 (interleukin 13), MME (membranemetallo-endopeptidase), TKT (transketolase), CXCR2 (chemokine (C-X-Cmotif) receptor 2), IGF1R (insulin-like growth factor 1 receptor), RARA(retinoic acid receptor, alpha), CREBBP (CREB binding protein), PTGS1(prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase andcyclooxygenase)), GALT (galactose-1-phosphate uridylyltransferase),CHRM1 (cholinergic receptor, muscarinic 1), ATXN1 (ataxin 1), PAWR(PRKC, apoptosis, WT1, regulator), NOTCH2 (Notch homolog 2(Drosophila)), M6PR (mannose-6-phosphate receptor (cation dependent)),CYP46A1 (cytochrome P450, family 46, subfamily A, polypeptide 1), CSNK1D (casein kinase 1, delta), MAPK14 (mitogen-activated protein kinase14), PRG2 (proteoglycan 2, bone marrow (natural killer cell activator,eosinophil granule major basic protein)), PRKCA (protein kinase C,alpha), L1 CAM (L1 cell adhesion molecule), CD40 (CD40 molecule, TNFreceptor superfamily member 5), NR1I2 (nuclear receptor subfamily 1,group I, member 2), JAG2 (jagged 2), CTNND1 (catenin(cadherin-associated protein), delta 1), CDH2 (cadherin 2, type 1,N-cadherin (neuronal)), CMA1 (chymase 1, mast cell), SORT1 (sortilin 1),DLK1 (delta-like 1 homolog (Drosophila)), THEM4 (thioesterasesuperfamily member 4), JUP (junction plakoglobin), CD46 (CD46 molecule,complement regulatory protein), CCL11 (chemokine (C-C motif) ligand 11),CAV3 (caveolin 3), RNASE3 (ribonuclease, RNase A family, 3 (eosinophilcationic protein)), HSPA8 (heat shock 70 kDa protein 8), CASP9 (caspase9, apoptosis-related cysteine peptidase), CYP3A4 (cytochrome P450,family 3, subfamily A, polypeptide 4), CCR3 (chemokine (C-C motif)receptor 3), TFAP2A (transcription factor AP-2 alpha (activatingenhancer binding protein 2 alpha)), SCP2 (sterol carrier protein 2),CDK4 (cyclin-dependent kinase 4), HIF1A (hypoxia inducible factor 1,alpha subunit (basic helix-loop-helix transcription factor)), TCF7L2(transcription factor 7-like 2 (T-cell specific, HMG-box)), IL1R2(interleukin 1 receptor, type II), B3GALTL (beta1,3-galactosyltransferase-like), MDM2 (Mdm2 p53 binding protein homolog(mouse)), RELA (v-rel reticuloendotheliosis viral oncogene homolog A(avian)), CASP7 (caspase 7, apoptosis-related cysteine peptidase), IDE(insulin-degrading enzyme), FABP4 (fatty acid binding protein 4,adipocyte), CASK (calcium/calmodulin-dependent serine protein kinase(MAGUK family)), ADCYAP1R1 (adenylate cyclase activating polypeptide 1(pituitary) receptor type I), ATF4 (activating transcription factor 4(tax-responsive enhancer element B67)), PDGFA (platelet-derived growthfactor alpha polypeptide), C21 or f33 (chromosome 21 open reading frame33), SCG5 (secretogranin V (7B2 protein)), RNF123 (ring finger protein123), NFKB1 (nuclear factor of kappa light polypeptide gene enhancer inB-cells 1), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogenehomolog 2, neuro/glioblastoma derived oncogene homolog (avian)), CAV1(caveolin 1, caveolae protein, 22 kDa), MMP7 (matrix metallopeptidase 7(matrilysin, uterine)), TGFA (transforming growth factor, alpha), RXRA(retinoid X receptor, alpha), STX1A (syntaxin 1A (brain)), PSMC4(proteasome (prosome, macropain) 26S subunit, ATPase, 4), P2RY2(purinergic receptor P2Y, G-protein coupled, 2), TNFRSF21 (tumornecrosis factor receptor superfamily, member 21), DLG1 (discs, largehomolog 1 (Drosophila)), NUMBL (numb homolog (Drosophila)-like), SPN(sialophorin), PLSCR1 (phospholipid scramblase 1), UBQLN2 (ubiquilin 2),UBQLN1 (ubiquilin 1), PCSK7 (proprotein convertase subtilisin/kexin type7), SPON1 (spondin 1, extracellular matrix protein), SILV (silverhomolog (mouse)), QPCT (glutaminyl-peptide cyclotransferase), HESS(hairy and enhancer of split 5 (Drosophila)), GCC1 (GRIP and coiled-coildomain containing 1), and any combination thereof.

The genetically modified animal or cell may comprise 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more disrupted chromosomal sequences encoding a proteinassociated with a secretase disorder and zero, 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more chromosomally integrated sequences encoding a disruptedprotein associated with a secretase disorder.

ALS

US Patent Publication No. 20110023144, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith amyotrophyic lateral sclerosis (ALS) disease. ALS is characterizedby the gradual steady degeneration of certain nerve cells in the braincortex, brain stem, and spinal cord involved in voluntary movement.

Motor neuron disorders and the proteins associated with these disordersare a diverse set of proteins that effect susceptibility for developinga motor neuron disorder, the presence of the motor neuron disorder, theseverity of the motor neuron disorder or any combination thereof. Thepresent disclosure comprises editing of any chromosomal sequences thatencode proteins associated with ALS disease, a specific motor neurondisorder. The proteins associated with ALS are typically selected basedon an experimental association of ALS-related proteins to ALS. Forexample, the production rate or circulating concentration of a proteinassociated with ALS may be elevated or depressed in a population withALS relative to a population without ALS. Differences in protein levelsmay be assessed using proteomic techniques including but not limited toWestern blot, immunohistochemical staining, enzyme linked immunosorbentassay (ELISA), and mass spectrometry. Alternatively, the proteinsassociated with ALS may be identified by obtaining gene expressionprofiles of the genes encoding the proteins using genomic techniquesincluding but not limited to DNA microarray analysis, serial analysis ofgene expression (SAGE), and quantitative real-time polymerase chainreaction (Q-PCR).

By way of non-limiting example, proteins associated with ALS include butare not limited to the following proteins: SOD1 superoxide dismutase 1,ALS3 amyotrophic lateral soluble sclerosis 3 SETX senataxin ALS5amyotrophic lateral sclerosis 5 FUS fused in sarcoma ALS7 amyotrophiclateral sclerosis 7 ALS2 amyotrophic lateral DPP6 Dipeptidyl-peptidase 6sclerosis 2 NEFH neurofilament, heavy PTGS1 prostaglandin-polypeptideendoperoxide synthase 1 SLC1A2 solute carrier family 1 TNFRSF10B tumornecrosis factor (glial high affinity receptor superfamily, glutamatetransporter), member 10b member 2 PRPH peripherin HSP90AA1 heat shockprotein 90 kDa alpha (cytosolic), class A member 1 GRIA2 glutamatereceptor, IFNG interferon, gamma ionotropic, AMPA 2 S 100B S100 calciumbinding FGF2 fibroblast growth factor 2 protein B AOX1 aldehyde oxidase1 CS citrate synthase TARDBP TAR DNA binding protein TXN thioredoxinRAPH1 Ras association MAP3K5 mitogen-activated protein (RaIGDS/AF-6) andkinase 5 pleckstrin homology domains 1 NBEAL1 neurobeachin-like 1 GPX1glutathione peroxidase 1 ICA1L islet cell autoantigen RAC1 ras-relatedC3 botulinum 1.69 kDa-like toxin substrate 1 MAPT microtubule-associatedITPR2 inositol 1,4,5-protein tau triphosphate receptor, type 2 ALS2CR4amyotrophic lateral GLS glutaminase sclerosis 2 (juvenile) chromosomeregion, candidate 4 ALS2CR8 amyotrophic lateral CNTFR ciliaryneurotrophic factor sclerosis 2 (juvenile) receptor chromosome region,candidate 8 ALS2CR11 amyotrophic lateral FOLH1 folate hydrolase 1sclerosis 2 (juvenile) chromosome region, candidate 11 FAM117B familywith sequence P4HB prolyl 4-hydroxylase, similarity 117, member B betapolypeptide CNTF ciliary neurotrophic factor SQSTM1 sequestosome 1STRADB STE20-related kinase NAIP NLR family, apoptosis adaptor betainhibitory protein YWHAQ tyrosine 3-SLC33A1 solute carrier family 33monooxygenase/tryptoph (acetyl-CoA transporter), an 5-monooxygenasemember 1 activation protein, theta polypeptide TRAK2 traffickingprotein, FIG. 4 homolog, SAC1 kinesin binding 2 lipid phosphatase domaincontaining NIF3L1 NIF3 NGG1 interacting INA internexin neuronal factor3-like 1 intermediate filament protein, alpha PARD3B par-3 partitioningCOX8A cytochrome c oxidase defective 3 homolog B subunit VIIIA CDK15cyclin-dependent kinase HECW1 HECT, C2 and WW 15 domain containing E3ubiquitin protein ligase 1 NOS1 nitric oxide synthase 1 MET metproto-oncogene SOD2 superoxide dismutase 2, HSPB1 heat shock 27 kDamitochondrial protein 1 NEFL neurofilament, light CTSB cathepsin Bpolypeptide ANG angiogenin, HSPA8 heat shock 70 kDa ribonuclease, RNaseA protein 8 family, 5 VAPB VAMP (vesicle-ESR1 estrogen receptor 1associated membrane protein)-associated protein B and C SNCA synuclein,alpha HGF hepatocyte growth factor CAT catalase ACTB actin, beta NEFMneurofilament, medium TH tyrosine hydroxylase polypeptide BCL2 B-cellCLL/lymphoma 2 FAS Fas (TNF receptor superfamily, member 6) CASP3caspase 3, apoptosis-CLU clusterin related cysteine peptidase SMN1survival of motor neuron G6PD glucose-6-phosphate 1, telomericdehydrogenase BAX BCL2-associated X HSF1 heat shock transcriptionprotein factor 1 RNF19A ring finger protein 19A JUN jun oncogeneALS2CR12 amyotrophic lateral HSPA5 heat shock 70 kDa sclerosis 2(juvenile) protein 5 chromosome region, candidate 12 MAPK14mitogen-activated protein IL10 interleukin 10 kinase 14 APEX1 APEXnuclease TXNRD1 thioredoxin reductase 1 (multifunctional DNA repairenzyme) 1 NOS2 nitric oxide synthase 2, TIMP1 TIMP metallopeptidaseinducible inhibitor 1 CASP9 caspase 9, apoptosis-XIAP X-linked inhibitorof related cysteine apoptosis peptidase GLG1 golgi glycoprotein 1 EPOerythropoietin VEGFA vascular endothelial ELN elastin growth factor AGDNF glial cell derived NFE2L2 nuclear factor (erythroid-neurotrophicfactor derived 2)-like 2 SLC6A3 solute carrier family 6 HSPA4 heat shock70 kDa (neurotransmitter protein 4 transporter, dopamine), member 3 APOEapolipoprotein E PSMB8 proteasome (prosome, macropain) subunit, betatype, 8 DCTN1 dynactin 1 TIMP3 TIMP metallopeptidase inhibitor 3 KIFAP3kinesin-associated SLC1A1 solute carrier family 1 protein 3(neuronal/epithelial high affinity glutamate transporter, system Xag),member 1 SMN2 survival of motor neuron CCNC cyclin C 2, centromeric MPP4membrane protein, STUB1 STIP1 homology and U-palmitoylated 4 boxcontaining protein 1 ALS2 amyloid beta (A4) PRDX6 peroxiredoxin 6precursor protein SYP synaptophysin CABIN1 calcineurin binding protein 1CASP1 caspase 1, apoptosis-GART phosphoribosylglycinami related cysteinede formyltransferase, peptidase phosphoribosylglycinami de synthetase,phosphoribosylaminoimi dazole synthetase CDK5 cyclin-dependent kinase 5ATXN3 ataxin 3 RTN4 reticulon 4 C1QB complement component 1, qsubcomponent, B chain VEGFC nerve growth factor HTT huntingtin receptorPARK7 Parkinson disease 7 XDH xanthine dehydrogenase GFAP glialfibrillary acidic MAP2 microtubule-associated protein protein 2 CYCScytochrome c, somatic FCGR3B Fc fragment of IgG, low affinity Mb, CCScopper chaperone for UBL5 ubiquitin-like 5 superoxide dismutase MMP9matrix metallopeptidase SLC18A3 solute carrier family 18 9 ((vesicularacetylcholine), member 3 TRPM7 transient receptor HSPB2 heat shock 27kDa potential cation channel, protein 2 subfamily M, member 7 AKT1 v-aktmurine thymoma DERL1 Derl-like domain family, viral oncogene homolog 1member 1 CCL2 chemokine (C-C motif) NGRN neugrin, neurite ligand 2outgrowth associated GSR glutathione reductase TPPP3 tubulinpolymerization-promoting protein family member 3 APAF1 apoptoticpeptidase BTBD10 BTB (POZ) domain activating factor 1 containing 10GLUD1 glutamate CXCR4 chemokine (C-X-C motif) dehydrogenase 1 receptor 4SLC1A3 solute carrier family 1 FLT1 fms-related tyrosine (glial highaffinity glutamate transporter), member 3 kinase 1 PON1 paraoxonase 1 ARandrogen receptor LIF leukemia inhibitory factor ERBB3 v-erb-b2erythroblastic leukemia viral oncogene homolog 3 LGALS1 lectin,galactoside-CD44 CD44 molecule binding, soluble, 1 TP53 tumor proteinp53 TLR3 toll-like receptor 3 GRIA1 glutamate receptor, GAPDHglyceraldehyde-3-ionotropic, AMPA 1 phosphate dehydrogenase GRIK1glutamate receptor, DES desmin ionotropic, kainate 1 CHAT cholineacetyltransferase FLT4 fms-related tyrosine kinase 4 CHMP2B chromatinmodifying BAG1 BCL2-associated protein 2B athanogene MT3 metallothionein3 CHRNA4 cholinergic receptor, nicotinic, alpha 4 GSS glutathionesynthetase BAK1 BCL2-antagonist/killer 1 KDR kinase insert domain GSTP1glutathione S-transferase receptor (a type III pi 1 receptor tyrosinekinase) OGG1 8-oxoguanine DNA IL6 interleukin 6 (interferon, glycosylasebeta 2).

The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredisrupted chromosomal sequences encoding a protein associated with ALSand zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomally integratedsequences encoding the disrupted protein associated with ALS. Preferredproteins associated with ALS include SOD1 (superoxide dismutase 1), ALS2(amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TARDNA binding protein), VAGFA (vascular endothelial growth factor A),VAGFB (vascular endothelial growth factor B), and VAGFC (vascularendothelial growth factor C), and any combination thereof.

Autism

US Patent Publication No. 20110023145, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith autism spectrum disorders (ASD). Autism spectrum disorders (ASDs)are a group of disorders characterized by qualitative impairment insocial interaction and communication, and restricted repetitive andstereotyped patterns of behavior, interests, and activities. The threedisorders, autism, Asperger syndrome (AS) and pervasive developmentaldisorder—not otherwise specified (PDD-NOS) are a continuum of the samedisorder with varying degrees of severity, associated intellectualfunctioning and medical conditions. ASDs are predominantly geneticallydetermined disorders with a heritability of around 90%.

US Patent Publication No. 20110023145 comprises editing of anychromosomal sequences that encode proteins associated with ASD which maybe applied to the CRISPR Cas system of the present invention. Theproteins associated with ASD are typically selected based on anexperimental association of the protein associated with ASD to anincidence or indication of an ASD. For example, the production rate orcirculating concentration of a protein associated with ASD may beelevated or depressed in a population having an ASD relative to apopulation lacking the ASD. Differences in protein levels may beassessed using proteomic techniques including but not limited to Westernblot, immunohistochemical staining, enzyme linked immunosorbent assay(ELISA), and mass spectrometry. Alternatively, the proteins associatedwith ASD may be identified by obtaining gene expression profiles of thegenes encoding the proteins using genomic techniques including but notlimited to DNA microarray analysis, serial analysis of gene expression(SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

Non limiting examples of disease states or disorders that may beassociated with proteins associated with ASD include autism, Aspergersyndrome (AS), pervasive developmental disorder—not otherwise specified(PDD-NOS), Rett's syndrome, tuberous sclerosis, phenylketonuria,Smith-Lemli-Opitz syndrome and fragile X syndrome. By way ofnon-limiting example, proteins associated with ASD include but are notlimited to the following proteins: ATP10C aminophospholipid-MET METreceptor transporting ATPase tyrosine kinase (ATP10C) BZRAP1 MGLUR5(GRM5) Metabotropic glutamate receptor 5 (MGLUR5) CDH10 Cadherin-10MGLUR6 (GRM6) Metabotropic glutamate receptor 6 (MGLUR6) CDH9 Cadherin-9NLGN1 Neuroligin-1 CNTN4 Contactin-4 NLGN2 Neuroligin-2 CNTNAP2Contactin-associated SEMA5A Neuroligin-3 protein-like 2 (CNTNAP2) DHCR77-dehydrocholesterol NLGN4X Neuroligin-4 X-reductase (DHCR7) linkedDOC2A Double C2-like domain-NLGN4Y Neuroligin-4 Y-containing proteinalpha linked DPP6 Dipeptidyl NLGN5 Neuroligin-5 aminopeptidase-likeprotein 6 EN2 engrailed 2 (EN2) NRCAM Neuronal cell adhesion molecule(NRCAM) MDGA2 fragile X mental retardation NRXN1 Neurexin-1 1 (MDGA2)FMR2 (AFF2) AF4/FMR2 family member 2 OR4M2 Olfactory receptor (AFF2) 4M2FOXP2 Forkhead box protein P2 OR4N4 Olfactory receptor (FOXP2) 4N4 FXR1Fragile X mental OXTR oxytocin receptor retardation, autosomal (OXTR)homolog 1 (FXR1) FXR2 Fragile X mental PAH phenylalanine retardation,autosomal hydroxylase (PAH) homolog 2 (FXR2) GABRA1 Gamma-aminobutyricacid PTEN Phosphatase and receptor subunit alpha-1 tensin homologue(GABRA1) (PTEN) GABRA5 GABAA (.gamma.-aminobutyric PTPRZ1 Receptor-typeacid) receptor alpha 5 tyrosine-protein subunit (GABRA5) phosphatasezeta (PTPRZ1) GABRB1 Gamma-aminobutyric acid RELN Reelin receptorsubunit beta-1 (GABRB1) GABRB3 GABAA (.gamma.-aminobutyric RPL10 60Sribosomal acid) receptor .beta.3 subunit protein L10 (GABRB3) GABRG1Gamma-aminobutyric acid SEMA5A Semaphorin-5A receptor subunit gamma-1(SEMA5A) (GABRG1) HIRIP3 HIRA-interacting protein 3 SEZ6L2 seizurerelated 6 homolog (mouse)-like 2 HOXA1 Homeobox protein Hox-A1 SHANK3SH3 and multiple (HOXA1) ankyrin repeat domains 3 (SHANK3) IL6Interleukin-6 SHBZRAP1 SH3 and multiple ankyrin repeat domains 3(SHBZRAP1) LAMB1 Laminin subunit beta-1 SLC6A4 Serotonin (LAMB1)transporter (SERT) MAPK3 Mitogen-activated protein TAS2R1 Taste receptorkinase 3 type 2 member 1 TAS2R1 MAZ Myc-associated zinc finger TSC1Tuberous sclerosis protein protein 1 MDGA2 MAM domain containing TSC2Tuberous sclerosis glycosylphosphatidylinositol protein 2 anchor 2(MDGA2) MECP2 Methyl CpG binding UBE3A Ubiquitin protein protein 2(MECP2) ligase E3A (UBE3A) MECP2 methyl CpG binding WNT2 Wingless-typeprotein 2 (MECP2) MMTV integration site family, member 2 (WNT2)

The identity of the protein associated with ASD whose chromosomalsequence is edited can and will vary. In preferred embodiments, theproteins associated with ASD whose chromosomal sequence is edited may bethe benzodiazapine receptor (peripheral) associated protein 1 (BZRAP1)encoded by the BZRAP1 gene, the AF4/FMR2 family member 2 protein (AFF2)encoded by the AFF2 gene (also termed MFR2), the fragile X mentalretardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene,the fragile X mental retardation autosomal homolog 2 protein (FXR2)encoded by the FXR2 gene, the MAM domain containingglycosylphosphatidylinositol anchor 2 protein (MDGA2) encoded by theMDGA2 gene, the methyl CpG binding protein 2 (MECP2) encoded by theMECP2 gene, the metabotropic glutamate receptor 5 (MGLURS) encoded bythe MGLUR5-1 gene (also termed GRM5), the neurexin 1 protein encoded bythe NRXN1 gene, or the semaphorin-5A protein (SEMA5A) encoded by theSEMA5A gene. In an exemplary embodiment, the genetically modified animalis a rat, and the edited chromosomal sequence encoding the proteinassociated with ASD is as listed below: BZRAP1 benzodiazapine receptorXM_002727789, (peripheral) associated XM_213427, protein 1 (BZRAP1)XM_002724533, XM_001081125 AFF2 (FMR2) AF4/FMR2 family member 2XM_219832, (AFF2) XM_001054673 FXR1 Fragile X mental NM_001012179retardation, autosomal homolog 1 (FXR1) FXR2 Fragile X mentalNM_001100647 retardation, autosomal homolog 2 (FXR2) MDGA2 MAM domaincontaining NM_199269 glycosylphosphatidylinositol anchor 2 (MDGA2) MECP2Methyl CpG binding NM_022673 protein 2 (MECP2) MGLUR5 Metabotropicglutamate NM_017012 (GRM5) receptor 5 (MGLUR5) NRXN1 Neurexin-1NM_021767 SEMA5A Semaphorin-5A (SEMA5A) NM_001107659.

Trinucleotide Repeat Expansion Disorders

US Patent Publication No. 20110016540, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith trinucleotide repeat expansion disorders. Trinucleotide repeatexpansion disorders are complex, progressive disorders that involvedevelopmental neurobiology and often affect cognition as well assensori-motor functions.

Trinucleotide repeat expansion proteins are a diverse set of proteinsassociated with susceptibility for developing a trinucleotide repeatexpansion disorder, the presence of a trinucleotide repeat expansiondisorder, the severity of a trinucleotide repeat expansion disorder orany combination thereof. Trinucleotide repeat expansion disorders aredivided into two categories determined by the type of repeat. The mostcommon repeat is the triplet CAG, which, when present in the codingregion of a gene, codes for the amino acid glutamine (Q). Therefore,these disorders are referred to as the polyglutamine (polyQ) disordersand comprise the following diseases: Huntington Disease (HD);Spinobulbar Muscular Atrophy (SBMA); Spinocerebellar Ataxias (SCA types1, 2, 3, 6, 7, and 17); and Dentatorubro-Pallidoluysian Atrophy (DRPLA).The remaining trinucleotide repeat expansion disorders either do notinvolve the CAG triplet or the CAG triplet is not in the coding regionof the gene and are, therefore, referred to as the non-polyglutaminedisorders. The non-polyglutamine disorders comprise Fragile X Syndrome(FRAXA); Fragile XE Mental Retardation (FRAXE); Friedreich Ataxia(FRDA); Myotonic Dystrophy (DM); and Spinocerebellar Ataxias (SCA types8, and 12).

The proteins associated with trinucleotide repeat expansion disordersare typically selected based on an experimental association of theprotein associated with a trinucleotide repeat expansion disorder to atrinucleotide repeat expansion disorder. For example, the productionrate or circulating concentration of a protein associated with atrinucleotide repeat expansion disorder may be elevated or depressed ina population having a trinucleotide repeat expansion disorder relativeto a population lacking the trinucleotide repeat expansion disorder.Differences in protein levels may be assessed using proteomic techniquesincluding but not limited to Western blot, immunohistochemical staining,enzyme linked immunosorbent assay (ELISA), and mass spectrometry.Alternatively, the proteins associated with trinucleotide repeatexpansion disorders may be identified by obtaining gene expressionprofiles of the genes encoding the proteins using genomic techniquesincluding but not limited to DNA microarray analysis, serial analysis ofgene expression (SAGE), and quantitative real-time polymerase chainreaction (Q-PCR).

Non-limiting examples of proteins associated with trinucleotide repeatexpansion disorders include AR (androgen receptor), FMR1 (fragile Xmental retardation 1), HTT (huntingtin), DMPK (dystrophiamyotonica-protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1(atrophin 1), FEN1 (flap structure-specific endonuclease 1), TNRC6A(trinucleotide repeat containing 6A), PABPN1 (poly(A) binding protein,nuclear 1), JPH3 (junctophilin 3), MED15 (mediator complex subunit 15),ATXN1 (ataxin 1), ATXN3 (ataxin 3), TBP (TATA box binding protein),CACNA1A (calcium channel, voltage-dependent, P/Q type, alpha 1Asubunit), ATXN80S (ATXN8 opposite strand (non-protein coding)), PPP2R2B(protein phosphatase 2, regulatory subunit B, beta), ATXN7 (ataxin 7),TNRC6B (trinucleotide repeat containing 6B), TNRC6C (trinucleotiderepeat containing 6C), CELF3 (CUGBP, Elav-like family member 3), MAB21L1(mab-21-like 1 (C. elegans)), MSH2 (mutS homolog 2, colon cancer,nonpolyposis type 1 (E. coli)), TMEM185A (transmembrane protein 185A),SIX5 (SIX homeobox 5), CNPY3 (canopy 3 homolog (zebrafish)), FRAXE(fragile site, folic acid type, rare, fra(X)(q28) E), GNB2 (guaninenucleotide binding protein (G protein), beta polypeptide 2), RPL14(ribosomal protein L14), ATXN8 (ataxin 8), INSR (insulin receptor), TTR(transthyretin), EP400 (E1A binding protein p400), GIGYF2 (GRB10interacting GYF protein 2), OGG1 (8-oxoguanine DNA glycosylase), STC1(stanniocalcin 1), CNDP1 (carnosine dipeptidase 1 (metallopeptidase M20family)), C10orf2 (chromosome 10 open reading frame 2), MAML3mastermind-like 3 (Drosophila), DKC1 (dyskeratosis congenita 1,dyskerin), PAXIP1 (PAX interacting (with transcription-activationdomain) protein 1), CASK (calcium/calmodulin-dependent serine proteinkinase (MAGUK family)), MAPT (microtubule-associated protein tau), SP1(Sp1 transcription factor), POLG (polymerase (DNA directed), gamma),AFF2 (AF4/FMR2 family, member 2), THBS1 (thrombospondin 1), TP53 (tumorprotein p53), ESR1 (estrogen receptor 1), CGGBP1 (CGG triplet repeatbinding protein 1), ABT1 (activator of basal transcription 1), KLK3(kallikrein-related peptidase 3), PRNP (prion protein), JUN (junoncogene), KCNN3 (potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 3), BAX (BCL2-associatedX protein), FRAXA (fragile site, folic acid type, rare, fra(X)(q27.3) A(macroorchidism, mental retardation)), KBTBD10 (kelch repeat and BTB(POZ) domain containing 10), MBNL1 (muscleblind-like (Drosophila)),RAD51 (RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)), NCOA3(nuclear receptor coactivator 3), ERDA1 (expanded repeat domain, CAG/CTG1), TSC1 (tuberous sclerosis 1), COMP (cartilage oligomeric matrixprotein), GCLC (glutamate-cysteine ligase, catalytic subunit), RRAD(Ras-related associated with diabetes), MSH3 (mutS homolog 3 (E. coli)),DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood group)),CTCF (CCCTC-binding factor (zinc finger protein)), CCND1 (cyclin D1),CLSPN (claspin homolog (Xenopus laevis)), MEF2A (myocyte enhancer factor2A), PTPRU (protein tyrosine phosphatase, receptor type, U), GAPDH(glyceraldehyde-3-phosphate dehydrogenase), TRIM22 (tripartitemotif-containing 22), WT1 (Wilms tumor 1), AHR (aryl hydrocarbonreceptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurineS-methyltransferase), NDP (Norrie disease (pseudoglioma)), ARX(aristaless related homeobox), MUS81 (MUS81 endonuclease homolog (S.cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (earlygrowth response 1), UNG (uracil-DNA glycosylase), NUMBL (numb homolog(Drosophila)-like), FABP2 (fatty acid binding protein 2, intestinal),EN2 (engrailed homeobox 2), CRYGC (crystallin, gamma C), SRP14 (signalrecognition particle 14 kDa (homologous Alu RNA binding protein)), CRYGB(crystallin, gamma B), PDCD1 (programmed cell death 1), HOXA1 (homeoboxA1), ATXN2L (ataxin 2-like), PMS2 (PMS2 postmeiotic segregationincreased 2 (S. cerevisiae)), GLA (galactosidase, alpha), CBL (Cas-Br-M(murine) ecotropic retroviral transforming sequence), FTH1 (ferritin,heavy polypeptide 1), IL12RB2 (interleukin 12 receptor, beta 2), OTX2(orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNAdirected), gamma 2, accessory subunit), DLX2 (distal-less homeobox 2),SIRPA (signal-regulatory protein alpha), OTX1 (orthodenticle homeobox1), AHRR (aryl-hydrocarbon receptor repressor), MANF (mesencephalicastrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein158 (gene/pseudogene)), and ENSG00000078687.

Preferred proteins associated with trinucleotide repeat expansiondisorders include HTT (Huntingtin), AR (androgen receptor), FXN(frataxin), Atxn3 (ataxin), Atxn1 (ataxin), Atxn2 (ataxin), Atxn7(ataxin), Atxn10 (ataxin), DMPK (dystrophia myotonica-protein kinase),Atn1 (atrophin 1), CBP (creb binding protein), VLDLR (very low densitylipoprotein receptor), and any combination thereof.

Treating Hearing Diseases

The present invention also contemplates delivering the CRISPR-Cas systemto one or both ears.

Researchers are looking into whether gene therapy could be used to aidcurrent deafness treatments—namely, cochlear implants. Deafness is oftencaused by lost or damaged hair cells that cannot relay signals toauditory neurons. In such cases, cochlear implants may be used torespond to sound and transmit electrical signals to the nerve cells. Butthese neurons often degenerate and retract from the cochlea as fewergrowth factors are released by impaired hair cells.

US patent application 20120328580 describes injection of apharmaceutical composition into the ear (e.g., auricularadministration), such as into the luminae of the cochlea (e.g., theScala media, Sc vestibulae, and Sc tympani), e.g., using a syringe,e.g., a single-dose syringe. For example, one or more of the compoundsdescribed herein can be administered by intratympanic injection (e.g.,into the middle ear), and/or injections into the outer, middle, and/orinner ear. Such methods are routinely used in the art, for example, forthe administration of steroids and antibiotics into human ears.Injection can be, for example, through the round window of the ear orthrough the cochlear capsule. Other inner ear administration methods areknown in the art (see, e.g., Salt and Plontke, Drug Discovery Today,10:1299-1306, 2005).

In another mode of administration, the pharmaceutical composition can beadministered in situ, via a catheter or pump. A catheter or pump can,for example, direct a pharmaceutical composition into the cochlearluminae or the round window of the ear and/or the lumen of the colon.Exemplary drug delivery apparatus and methods suitable for administeringone or more of the compounds described herein into an ear, e.g., a humanear, are described by McKenna et al., (U.S. Publication No.2006/0030837) and Jacobsen et al., (U.S. Pat. No. 7,206,639). In someembodiments, a catheter or pump can be positioned, e.g., in the ear(e.g., the outer, middle, and/or inner ear) of a patient during asurgical procedure. In some embodiments, a catheter or pump can bepositioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear)of a patient without the need for a surgical procedure.

Alternatively or in addition, one or more of the compounds describedherein can be administered in combination with a mechanical device suchas a cochlear implant or a hearing aid, which is worn in the outer ear.An exemplary cochlear implant that is suitable for use with the presentinvention is described by Edge et al., (U.S. Publication No.2007/0093878).

In some embodiments, the modes of administration described above may becombined in any order and can be simultaneous or interspersed.

Alternatively or in addition, the present invention may be administeredaccording to any of the Food and Drug Administration approved methods,for example, as described in CDER Data Standards Manual, version number004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm).

In general, the cell therapy methods described in US patent application20120328580 can be used to promote complete or partial differentiationof a cell to or towards a mature cell type of the inner ear (e.g., ahair cell) in vitro. Cells resulting from such methods can then betransplanted or implanted into a patient in need of such treatment. Thecell culture methods required to practice these methods, includingmethods for identifying and selecting suitable cell types, methods forpromoting complete or partial differentiation of selected cells, methodsfor identifying complete or partially differentiated cell types, andmethods for implanting complete or partially differentiated cells aredescribed below.

Cells suitable for use in the present invention include, but are notlimited to, cells that are capable of differentiating completely orpartially into a mature cell of the inner ear, e.g., a hair cell (e.g.,an inner and/or outer hair cell), when contacted, e.g., in vitro, withone or more of the compounds described herein. Exemplary cells that arecapable of differentiating into a hair cell include, but are not limitedto stem cells (e.g., inner ear stem cells, adult stem cells, bone marrowderived stem cells, embryonic stem cells, mesenchymal stem cells, skinstem cells, iPS cells, and fat derived stem cells), progenitor cells(e.g., inner ear progenitor cells), support cells (e.g., Deiters' cells,pillar cells, inner phalangeal cells, tectal cells and Hensen's cells),and/or germ cells. The use of stem cells for the replacement of innerear sensory cells is described in Li et al., (U.S. Publication No.2005/0287127) and Li et al., (U.S. patent Ser. No. 11/953,797). The useof bone marrow derived stem cells for the replacement of inner earsensory cells is described in Edge et al., PCT/US2007/084654. iPS cellsare described, e.g., at Takahashi et al., Cell, Volume 131, Issue 5,Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006);Okita et al., Nature 448, 260-262 (2007); Yu, J. et al., Science318(5858):1917-1920 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106(2008); and Zaehres and Scholer, Cell 131(5):834-835 (2007). Suchsuitable cells can be identified by analyzing (e.g., qualitatively orquantitatively) the presence of one or more tissue specific genes. Forexample, gene expression can be detected by detecting the proteinproduct of one or more tissue-specific genes. Protein detectiontechniques involve staining proteins (e.g., using cell extracts or wholecells) using antibodies against the appropriate antigen. In this case,the appropriate antigen is the protein product of the tissue-specificgene expression. Although, in principle, a first antibody (i.e., theantibody that binds the antigen) can be labeled, it is more common (andimproves the visualization) to use a second antibody directed againstthe first (e.g., an anti-IgG). This second antibody is conjugated eitherwith fluorochromes, or appropriate enzymes for colorimetric reactions,or gold beads (for electron microscopy), or with the biotin-avidinsystem, so that the location of the primary antibody, and thus theantigen, can be recognized.

The CRISPR Cas molecules of the present invention may be delivered tothe ear by direct application of pharmaceutical composition to the outerear, with compositions modified from US Published application,20110142917. In some embodiments the pharmaceutical composition isapplied to the ear canal. Delivery to the ear may also be referred to asaural or otic delivery.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed, (see, forexample, Shen et al FEBS Let. 2003, 539:111-114; Xia et al., Nat.Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision. 2003, 9:210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis etal., Nat. Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11:2717-2724) and may be applied to the present invention. siRNA hasrecently been successfully used for inhibition of gene expression inprimates (see for example. Tolentino et al., Retina 24(4):660 which mayalso be applied to the present invention.

Qi et al. discloses methods for efficient siRNA transfection to theinner ear through the intact round window by a novel proteidic deliverytechnology which may be applied to the nucleic acid-targeting system ofthe present invention (see, e.g., Qi et al., Gene Therapy (2013), 1-9).In particular, a TAT double stranded RNA-binding domains (TAT-DRBDs),which can transfect Cy3-labeled siRNA into cells of the inner ear,including the inner and outer hair cells, crista ampullaris, maculautriculi and macula sacculi, through intact round-window permeation wassuccessful for delivering double stranded siRNAs in vivo for treatingvarious inner ear ailments and preservation of hearing function. About40 μl of 10 mM RNA may be contemplated as the dosage for administrationto the ear.

According to Rejali et al. (Hear Res. 2007 June; 228(1-2):180-7),cochlear implant function can be improved by good preservation of thespiral ganglion neurons, which are the target of electrical stimulationby the implant and brain derived neurotrophic factor (BDNF) haspreviously been shown to enhance spiral ganglion survival inexperimentally deafened ears. Rejali et al. tested a modified design ofthe cochlear implant electrode that includes a coating of fibroblastcells transduced by a viral vector with a BDNF gene insert. Toaccomplish this type of ex vivo gene transfer, Rejali et al. transducedguinea pig fibroblasts with an adenovirus with a BDNF gene cassetteinsert, and determined that these cells secreted BDNF and then attachedBDNF-secreting cells to the cochlear implant electrode via an agarosegel, and implanted the electrode in the scala tympani. Rejali et al.determined that the BDNF expressing electrodes were able to preservesignificantly more spiral ganglion neurons in the basal turns of thecochlea after 48 days of implantation when compared to controlelectrodes and demonstrated the feasibility of combining cochlearimplant therapy with ex vivo gene transfer for enhancing spiral ganglionneuron survival. Such a system may be applied to the nucleicacid-targeting system of the present invention for delivery to the ear.

Mukherjea et al. (Antioxidants & Redox Signaling, Volume 13, Number 5,2010) document that knockdown of NOX3 using short interfering (si) RNAabrogated cisplatin ototoxicity, as evidenced by protection of OHCs fromdamage and reduced threshold shifts in auditory brainstem responses(ABRs). Different doses of siNOX3 (0.3, 0.6, and 0.9 μg) wereadministered to rats and NOX3 expression was evaluated by real timeRT-PCR. The lowest dose of NOX3 siRNA used (0.3 μg) did not show anyinhibition of NOX3 mRNA when compared to transtympanic administration ofscrambled siRNA or untreated cochleae. However, administration of thehigher doses of NOX3 siRNA (0.6 and 0.9 μg) reduced NOX3 expressioncompared to control scrambled siRNA. Such a system may be applied to theCRISPR Cas system of the present invention for transtympanicadministration with a dosage of about 2 mg to about 4 mg of CRISPR Casfor administration to a human.

Jung et al. (Molecular Therapy, vol. 21 no. 4, 834-841 April 2013)demonstrate that Hes5 levels in the utricle decreased after theapplication of siRNA and that the number of hair cells in these utricleswas significantly larger than following control treatment. The datasuggest that siRNA technology may be useful for inducing repair andregeneration in the inner ear and that the Notch signaling pathway is apotentially useful target for specific gene expression inhibition. Junget al. injected 8 μg of Hes5 siRNA in 2 μl volume, prepared by addingsterile normal saline to the lyophilized siRNA to a vestibularepithelium of the ear. Such a system may be applied to the nucleicacid-targeting system of the present invention for administration to thevestibular epithelium of the ear with a dosage of about 1 to about 30 mgof CRISPR Cas for administration to a human.

Gene Targeting in Non-Dividing Cells (Neurones & Muscle)

Non-dividing (especially non-dividing, fully differentiated) cell typespresent issues for gene targeting or genome engineering, for examplebecause homologous recombination (HR) is generally suppressed in the G1cell-cycle phase. However, while studying the mechanisms by which cellscontrol normal DNA repair systems, Durocher discovered a previouslyunknown switch that keeps HR “off” in non-dividing cells and devised astrategy to toggle this switch back on. Orthwein et al. (DanielDurocher's lab at the Mount Sinai Hospital in Ottawa, Canada) recentlyreported (Nature 16142, published online 9 Dec. 2015) have shown thatthe suppression of HR can be lifted and gene targeting successfullyconcluded in both kidney (293T) and osteosarcoma (U205) cells. Tumorsuppressors, BRCA1, PALB2 and BRAC2 are known to promote DNA DSB repairby HR. They found that formation of a complex of BRCA1 with PALB2-BRAC2is governed by a ubiquitin site on PALB2, such that action on the siteby an E3 ubiquitin ligase. This E3 ubiquitin ligase is composed of KEAP1(a PALB2-interacting protein) in complex with cullin-3 (CUL3)-RBX1.PALB2 ubiquitylation suppresses its interaction with BRCA1 and iscounteracted by the deubiquitylase USP11, which is itself under cellcycle control. Restoration of the BRCA1-PALB2 interaction combined withthe activation of DNA-end resection is sufficient to induce homologousrecombination in G1, as measured by a number of methods including aCRISPR-Cas9-based gene-targeting assay directed at USP11 or KEAP1(expressed from a pX459 vector). However, when the BRCA1-PALB2interaction was restored in resection-competent G1 cells using eitherKEAP1 depletion or expression of the PALB2-KR mutant, a robust increasein gene-targeting events was detected.

Thus, reactivation of HR in cells, especially non-dividing, fullydifferentiated cell types is preferred, in some embodiments. In someembodiments, promotion of the BRCA1-PALB2 interaction is preferred insome embodiments. In some embodiments, the target cell is a non-dividingcell. In some embodiments, the target cell is a neurone or muscle cell.In some embodiments, the target cell is targeted in vivo. In someembodiments, the cell is in G1 and HR is suppressed. In someembodiments, use of KEAP1 depletion, for example inhibition ofexpression of KEAP1 activity, is preferred. KEAP1 depletion may beachieved through siRNA, for example as shown in Orthwein et al.Alternatively, expression of the PALB2-KR mutant (lacking all eight Lysresidues in the BRCA1-interaction domain is preferred, either incombination with KEAP1 depletion or alone. PALB2-KR interacts with BRCA1irrespective of cell cycle position. Thus, promotion or restoration ofthe BRCA1-PALB2 interaction, especially in G1 cells, is preferred insome embodiments, especially where the target cells are non-dividing, orwhere removal and return (ex vivo gene targeting) is problematic, forexample neurone or muscle cells. KEAP1 siRNA is available fromThermoFischer. In some embodiments, a BRCA1-PALB2 complex may bedelivered to the G1 cell. In some embodiments, PALB2 deubiquitylationmay be promoted for example by increased expression of thedeubiquitylase USP11, so it is envisaged that a construct may beprovided to promote or up-regulate expression or activity of thedeubiquitylase USP11.

Treating Diseases of the Eye

The present invention also contemplates delivering the CRISPR-Cas systemto one or both eyes.

In particular embodiments of the invention, the CRISPR-Cas system may beused to correct ocular defects that arise from several genetic mutationsfurther described in Genetic Diseases of the Eye, Second Edition, editedby Elias I. Traboulsi, Oxford University Press, 2012.

For administration to the eye, lentiviral vectors, in particular equineinfectious anemia viruses (EIAV) are particularly preferred.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285, Published online 21 Nov. 2005 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/jgm.845). The vectors arecontemplated to have cytomegalovirus (CMV) promoter driving expressionof the target gene. Intracameral, subretinal, intraocular andintravitreal injections are all contemplated (see, e.g., Balagaan, JGene Med 2006; 8: 275-285, Published online 21 Nov. 2005 in WileyInterScience (www.interscience.wiley.com). DOI: 10.1002/jgm. 845).Intraocular injections may be performed with the aid of an operatingmicroscope. For subretinal and intravitreal injections, eyes may beprolapsed by gentle digital pressure and fundi visualised using acontact lens system consisting of a drop of a coupling medium solutionon the cornea covered with a glass microscope slide coverslip. Forsubretinal injections, the tip of a 10-mm 34-gauge needle, mounted on a5-μl Hamilton syringe may be advanced under direct visualisation throughthe superior equatorial sclera tangentially towards the posterior poleuntil the aperture of the needle was visible in the subretinal space.Then, 2 μl of vector suspension may be injected to produce a superiorbullous retinal detachment, thus confirming subretinal vectoradministration. This approach creates a self-sealing sclerotomy allowingthe vector suspension to be retained in the subretinal space until it isabsorbed by the RPE, usually within 48 h of the procedure. Thisprocedure may be repeated in the inferior hemisphere to produce aninferior retinal detachment. This technique results in the exposure ofapproximately 70% of neurosensory retina and RPE to the vectorsuspension. For intravitreal injections, the needle tip may be advancedthrough the sclera 1 mm posterior to the corneoscleral limbus and 2 μlof vector suspension injected into the vitreous cavity. For intracameralinjections, the needle tip may be advanced through a corneosclerallimbal paracentesis, directed towards the central cornea, and 2 μl ofvector suspension may be injected. For intracameral injections, theneedle tip may be advanced through a corneoscleral limbal paracentesis,directed towards the central cornea, and 2 μl of vector suspension maybe injected. These vectors may be injected at titres of either1.0-1.4×10¹⁰ or 1.0-1.4×10⁹ transducing units (TU)/ml.

In another embodiment, RetinoStat®, an equine infectious anemiavirus-based lentiviral gene therapy vector that expresses angiostaticproteins endostain and angiostatin that is delivered via a subretinalinjection for the treatment of the web form of age-related maculardegeneration is also contemplated (see, e.g., Binley et al., HUMAN GENETHERAPY 23:980-991 (September 2012)). Such a vector may be modified forthe CRISPR-Cas system of the present invention. Each eye may be treatedwith either RetinoStat® at a dose of 1.1×10⁵ transducing units per eye(TU/eye) in a total volume of 100 μl.

In another embodiment, an E1-, partial E3-, E4-deleted adenoviral vectormay be contemplated for delivery to the eye. Twenty-eight patients withadvanced neovascular agerelated macular degeneration (AMD) were given asingle intravitreous injection of an E1-, partial E3-, E4-deletedadenoviral vector expressing human pigment ep-ithelium-derived factor(AdPEDF.ll) (see, e.g., Campochiaro et al., Human Gene Therapy17:167-176 (February 2006)). Doses ranging from 10⁶ to 10^(9.5) particleunits (PU) were investigated and there were no serious adverse eventsrelated to AdPEDF.ll and no dose-limiting toxicities (see, e.g.,Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)).Adenoviral vectormediated ocular gene transfer appears to be a viableapproach for the treatment of ocular disorders and could be applied tothe CRISPR Cas system.

In another embodiment, the sd-rxRNA® system of RXi Pharmaceuticals maybe used/and or adapted for delivering CRISPR Cas to the eye. In thissystem, a single intravitreal administration of 3 μg of sd-rxRNA resultsin sequence-specific reduction of PPIB mRNA levels for 14 days. Thesd-rxRNA® system may be applied to the nucleic acid-targeting system ofthe present invention, contemplating a dose of about 3 to 20 mg ofCRISPR administered to a human.

Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 April2011) describes adeno-associated virus (AAV) vectors to deliver an RNAinterference (RNAi)-based rhodopsin suppressor and a codon-modifiedrhodopsin replacement gene resistant to suppression due to nucleotidealterations at degenerate positions over the RNAi target site. Aninjection of either 6.0×10⁸ vp or 1.8×10¹⁰ vp AAV were subretinallyinjected into the eyes by Millington-Ward et al. The AAV vectors ofMillington-Ward et al. may be applied to the CRISPR Cas system of thepresent invention, contemplating a dose of about 2×10¹¹ to about 6×10¹³vp administered to a human.

Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also relates to invivo directed evolution to fashion an AAV vector that delivers wild-typeversions of defective genes throughout the retina after noninjuriousinjection into the eyes' vitreous humor. Dalkara describes a 7 merpeptide display library and an AAV library constructed by DNA shufflingof cap genes from AAV1, 2, 4, 5, 6, 8, and 9. The rcAAV libraries andrAAV vectors expressing GFP under a CAG or Rho promoter were packagedand deoxyribonuclease-resistant genomic titers were obtained throughquantitative PCR. The libraries were pooled, and two rounds of evolutionwere performed, each consisting of initial library diversificationfollowed by three in vivo selection steps. In each such step, P30rho-GFP mice were intravitreally injected with 2 ml ofiodixanol-purified, phosphate-buffered saline (PBS)-dialyzed librarywith a genomic titer of about 1×10¹² vg/ml. The AAV vectors of Dalkaraet al. may be applied to the nucleic acid-targeting system of thepresent invention, contemplating a dose of about 1×10¹⁵ to about 1×10¹⁶vg/ml administered to a human.

In a particular embodiment, the rhodopsin gene may be targeted for thetreatment of retinitis pigmentosa (RP), wherein the system of US PatentPublication No. 20120204282 assigned to Sangamo BioSciences, Inc. may bemodified in accordance of the CRISPR Cas system of the presentinvention.

In another embodiment, the methods of US Patent Publication No.20130183282 assigned to Cellectis, which is directed to methods ofcleaving a target sequence from the human rhodopsin gene, may also bemodified to the nucleic acid-targeting system of the present invention.

US Patent Publication No. 20130202678 assigned to Academia Sinicarelates to methods for treating retinopathies and sight-threateningophthalmologic disorders relating to delivering of the Puf-A gene (whichis expressed in retinal ganglion and pigmented cells of eye tissues anddisplays a unique anti-apoptotic activity) to the sub-retinal orintravitreal space in the eye. In particular, desirable targets arezgc:193933, prdm1a, spata2, tex10, rbb4, ddx3, zp2.2, Blimp-1 and HtrA2,all of which may be targeted by the nucleic acid-targeting system of thepresent invention.

Wu (Cell Stem Cell, 13:659-62, 2013) designed a guide RNA that led Cas9to a single base pair mutation that causes cataracts in mice, where itinduced DNA cleavage. Then using either the other wild-type allele oroligos given to the zygotes repair mechanisms corrected the sequence ofthe broken allele and corrected the cataract-causing genetic defect inmutant mouse.

US Patent Publication No. 20120159653, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith macular degeration (MD). Macular degeneration (MD) is the primarycause of visual impairment in the elderly, but is also a hallmarksymptom of childhood diseases such as Stargardt disease, Sorsby fundus,and fatal childhood neurodegenerative diseases, with an age of onset asyoung as infancy. Macular degeneration results in a loss of vision inthe center of the visual field (the macula) because of damage to theretina. Currently existing animal models do not recapitulate majorhallmarks of the disease as it is observed in humans. The availableanimal models comprising mutant genes encoding proteins associated withMD also produce highly variable phenotypes, making translations to humandisease and therapy development problematic.

One aspect of US Patent Publication No. 20120159653 relates to editingof any chromosomal sequences that encode proteins associated with MDwhich may be applied to the nucleic acid-targeting system of the presentinvention. The proteins associated with MD are typically selected basedon an experimental association of the protein associated with MD to anMD disorder. For example, the production rate or circulatingconcentration of a protein associated with MD may be elevated ordepressed in a population having an MD disorder relative to a populationlacking the MD disorder. Differences in protein levels may be assessedusing proteomic techniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the proteins associated with MDmay be identified by obtaining gene expression profiles of the genesencoding the proteins using genomic techniques including but not limitedto DNA microarray analysis, serial analysis of gene expression (SAGE),and quantitative real-time polymerase chain reaction (Q-PCR).

By way of non-limiting example, proteins associated with MD include butare not limited to the following proteins: (ABCA4) ATP-binding cassette,sub-family A (ABC1), member 4 ACHM1 achromatopsia (rod monochromacy) 1ApoE Apolipoprotein E (ApoE) C1QTNF5 (CTRP5) C1q and tumor necrosisfactor related protein 5 (C1QTNF5) C2 Complement component 2 (C2) C3Complement components (C3) CCL2 Chemokine (C-C motif) Ligand 2 (CCL2)CCR2 Chemokine (C-C motif) receptor 2 (CCR2) CD36 Cluster ofDifferentiation 36 CFB Complement factor B CFH Complement factor CFH HCFHR1 complement factor H-related 1 CFHR3 complement factor H-related 3CNGB3 cyclic nucleotide gated channel beta 3 CP ceruloplasmin (CP) CRP Creactive protein (CRP) CST3 cystatin C or cystatin 3 (CST3) CTSDCathepsin D (CTSD) CX3CR1 chemokine (C-X3-C motif) receptor 1 ELOVL4Elongation of very long chain fatty acids 4 ERCC6 excision repaircrosscomplementing rodent repair deficiency, complementation group 6FBLN5 Fibulin-5 FBLN5 Fibulin 5 FBLN6 Fibulin 6 FSCN2 fascin (FSCN2)HMCN1 Hemicentrin 1 HMCN1 hemicentin 1 HTRA1 HtrA serine peptidase 1(HTRA1) HTRA1 HtrA serine peptidase 1 IL-6 Interleukin 6 IL-8Interleukin 8 LOC387715 Hypothetical protein PLEKHA1 Pleckstrin homologydomaincontaining family A member 1 (PLEKHA1) PROM1 Prominin 1 (PROM1 orCD133) PRPH2 Peripherin-2 RPGR retinitis pigmentosa GTPase regulatorSERPING1 serpin peptidase inhibitor, clade G, member 1 (C1-inhibitor)TCOF1 Treacle TIMP3 Metalloproteinase inhibitor 3 (TIMP3) TLR3 Toll-likereceptor 3.

The identity of the protein associated with MD whose chromosomalsequence is edited can and will vary. In preferred embodiments, theproteins associated with MD whose chromosomal sequence is edited may bethe ATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4)encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded bythe APOE gene, the chemokine (C-C motif) Ligand 2 protein (CCL2) encodedby the CCL2 gene, the chemokine (C-C motif) receptor 2 protein (CCR2)encoded by the CCR2 gene, the ceruloplasmin protein (CP) encoded by theCP gene, the cathepsin D protein (CTSD) encoded by the CTSD gene, or themetalloproteinase inhibitor 3 protein (TIMP3) encoded by the TIMP3 gene.In an exemplary embodiment, the genetically modified animal is a rat,and the edited chromosomal sequence encoding the protein associated withMD may be: (ABCA4) ATPbinding cassette, NM_000350 sub-family A (ABC1),member 4 APOE Apolipoprotein E NM_138828 (APOE) CCL2 Chemokine (C-CNM_031530 motif) Ligand 2 (CCL2) CCR2 Chemokine (C-C NM_021866 motif)receptor 2 (CCR2) CP ceruloplasmin (CP) NM_012532 CTSD Cathepsin D(CTSD) NM_134334 TIMP3 Metalloproteinase NM_012886 inhibitor 3 (TIMP3)The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7 or more disruptedchromosomal sequences encoding a protein associated with MD and zero, 1,2, 3, 4, 5, 6, 7 or more chromosomally integrated sequences encoding thedisrupted protein associated with MD.

The edited or integrated chromosomal sequence may be modified to encodean altered protein associated with MD. Several mutations in MD-relatedchromosomal sequences have been associated with MD. Non-limitingexamples of mutations in chromosomal sequences associated with MDinclude those that may cause MD including in the ABCR protein, E471K(i.e. glutamate at position 471 is changed to lysine), R1129L (i.e.arginine at position 1129 is changed to leucine), T1428M (i.e. threonineat position 1428 is changed to methionine), R1517S (i.e. arginine atposition 1517 is changed to serine), I1562T (i.e. isoleucine at position1562 is changed to threonine), and G1578R (i.e. glycine at position 1578is changed to arginine); in the CCR2 protein, V64I (i.e. valine atposition 192 is changed to isoleucine); in CP protein, G969B (i.e.glycine at position 969 is changed to asparagine or aspartate); in TIMP3protein, S156C (i.e. serine at position 156 is changed to cysteine),G166C (i.e. glycine at position 166 is changed to cysteine), G167C (i.e.glycine at position 167 is changed to cysteine), Y168C (i.e. tyrosine atposition 168 is changed to cysteine), 5170C (i.e. serine at position 170is changed to cysteine), Y172C (i.e. tyrosine at position 172 is changedto cysteine) and S181C (i.e. serine at position 181 is changed tocysteine). Other associations of genetic variants in MD-associated genesand disease are known in the art.

CRISPR systems are useful to correct diseases resulting from autosomaldominant genes. For example, CRISPR/Cas9 was used to remove an autosomaldominant gene that causes receptor loss in the eye. Bakondi, B. et al.,In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in theS334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa.Molecular Therapy, 2015; DOI: 10.1038/mt.2015.220.

Treating Circulatory and Muscular Diseases

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. Cpf1 effector protein systems, to the heart. Forthe heart, a myocardium tropic adena-associated virus (AAVM) ispreferred, in particular AAVM41 which showed preferential gene transferin the heart (see, e.g., Lin-Yanga et al., PNAS, Mar. 10, 2009, vol.106, no. 10). Administration may be systemic or local. A dosage of about1-10×10¹⁴ vector genomes are contemplated for systemic administration.See also, e.g., Eulalio et al. (2012) Nature 492: 376 and Somasuntharamet al. (2013) Biomaterials 34: 7790.

For example, US Patent Publication No. 20110023139, describes use ofzinc finger nucleases to genetically modify cells, animals and proteinsassociated with cardiovascular disease. Cardiovascular diseasesgenerally include high blood pressure, heart attacks, heart failure, andstroke and TIA. Any chromosomal sequence involved in cardiovasculardisease or the protein encoded by any chromosomal sequence involved incardiovascular disease may be utilized in the methods described in thisdisclosure. The cardiovascular-related proteins are typically selectedbased on an experimental association of the cardiovascular-relatedprotein to the development of cardiovascular disease. For example, theproduction rate or circulating concentration of a cardiovascular-relatedprotein may be elevated or depressed in a population having acardiovascular disorder relative to a population lacking thecardiovascular disorder. Differences in protein levels may be assessedusing proteomic techniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the cardiovascular-relatedproteins may be identified by obtaining gene expression profiles of thegenes encoding the proteins using genomic techniques including but notlimited to DNA microarray analysis, serial analysis of gene expression(SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

By way of example, the chromosomal sequence may comprise, but is notlimited to, IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase),TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin)synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1),ABCG8 (ATP-binding cassette, sub-family G (WHITE), member 8), CTSK(cathepsin K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)),KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11),INS (insulin), CRP (C-reactive protein, pentraxin-related), PDGFRB(platelet-derived growth factor receptor, beta polypeptide), CCNA2(cyclin A2), PDGFB (platelet-derived growth factor beta polypeptide(simian sarcoma viral (v-sis) oncogene homolog)), KCNJ5 (potassiuminwardly-rectifying channel, subfamily J, member 5), KCNN3 (potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 3), CAPN10 (calpain 10), PTGES (prostaglandin E synthase), ADRA2B(adrenergic, alpha-2B-, receptor), ABCG5 (ATP-binding cassette,sub-family G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calpain5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C(mex-3 homolog C (C. elegans)), ACE angiotensin I converting enzyme(peptidyl-dipeptidase A) 1), TNF (tumor necrosis factor (TNFsuperfamily, member 2)), IL6 (interleukin 6 (interferon, beta 2)), STN(statin), SERPINE1 (serpin peptidase inhibitor, clade E (nexin,plasminogen activator inhibitor type 1), member 1), ALB (albumin),ADIPOQ (adiponectin, C1Q and collagen domain containing), APOB(apolipoprotein B (including Ag(x) antigen)), APOE (apolipoprotein E),LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)),APOA1 (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (natriureticpeptide precursor B), NOS3 (nitric oxide synthase 3 (endothelial cell)),PPARG (peroxisome proliferator-activated receptor gamma), PLAT(plasminogen activator, tissue), PTGS2 (prostaglandin-endoperoxidesynthase 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP(cholesteryl ester transfer protein, plasma), AGTR1 (angiotensin IIreceptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme Areductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE(selectin E), REN (renin), PPARA (peroxisome proliferator-activatedreceptor alpha), PON1 (paraoxonase 1), KNG1 (kininogen 1), CCL2(chemokine (C-C motif) ligand 2), LPL (lipoprotein lipase), VWF (vonWillebrand factor), F2 (coagulation factor II (thrombin)), ICAM1(intercellular adhesion molecule 1), TGFB1 (transforming growth factor,beta 1), NPPA (natriuretic peptide precursor A), IL10 (interleukin 10),EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1(vascular cell adhesion molecule 1), IFNG (interferon, gamma), LPA(lipoprotein, Lp(a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1),MAPK1 (mitogen-activated protein kinase 1), HP (haptoglobin), F3(coagulation factor III (thromboplastin, tissue factor)), CST3 (cystatinC), COG2 (component of oligomeric golgi complex 2), MMP9 (matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase)), SERPINC1 (serpin peptidase inhibitor, clade C(antithrombin), member 1), F8 (coagulation factor VIII, procoagulantcomponent), HMOX1 (heme oxygenase (decycling) 1), APOC3 (apolipoproteinC-III), IL8 (interleukin 8), PROK1 (prokineticin 1), CBS(cystathionine-beta-synthase), NOS2 (nitric oxide synthase 2,inducible), TLR4 (toll-like receptor 4), SELP (selectin P (granulemembrane protein 140 kDa, antigen CD62)), ABCA1 (ATP-binding cassette,sub-family A (ABC1), member 1), AGT (angiotensinogen (serpin peptidaseinhibitor, clade A, member 8)), LDLR (low density lipoprotein receptor),GPT (glutamic-pyruvate transaminase (alanine aminotransferase)), VEGFA(vascular endothelial growth factor A), NR3C2 (nuclear receptorsubfamily 3, group C, member 2), IL18 (interleukin 18(interferon-gamma-inducing factor)), NOS1 (nitric oxide synthase 1(neuronal)), NR3C1 (nuclear receptor subfamily 3, group C, member 1(glucocorticoid receptor)), FGB (fibrinogen beta chain), HGF (hepatocytegrowth factor (hepapoietin A; scatter factor)), ILIA (interleukin 1,alpha), RETN (resistin), AKT1 (v-akt murine thymoma viral oncogenehomolog 1), LIPC (lipase, hepatic), HSPD1 (heat shock 60 kDa protein 1(chaperonin)), MAPK14 (mitogen-activated protein kinase 14), SPP1(secreted phosphoprotein 1), ITGB3 (integrin, beta 3 (plateletglycoprotein 111a, antigen CD61)), CAT (catalase), UTS2 (urotensin 2),THBD (thrombomodulin), F10 (coagulation factor X), CP (ceruloplasmin(ferroxidase)), TNFRSF11B (tumor necrosis factor receptor superfamily,member 11b), EDNRA (endothelin receptor type A), EGFR (epidermal growthfactor receptor (erythroblastic leukemia viral (v-erb-b) oncogenehomolog, avian)), MMP2 (matrix metallopeptidase 2 (gelatinase A, 72 kDagelatinase, 72 kDa type IV collagenase)), PLG (plasminogen), NPY(neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8(mitogen-activated protein kinase 8), MYC (v-myc myelocytomatosis viraloncogene homolog (avian)), FN1 (fibronectin 1), CMA1 (chymase 1, mastcell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotidebinding protein (G protein), beta polypeptide 3), ADRB2 (adrenergic,beta-2-, receptor, surface), APOA5 (apolipoprotein A-V), SOD2(superoxide dismutase 2, mitochondrial), F5 (coagulation factor V(proaccelerin, labile factor)), VDR (vitamin D (1,25-dihydroxyvitaminD3) receptor), ALOX5 (arachidonate 5-lipoxygenase), HLA-DRB1 (majorhistocompatibility complex, class II, DR beta 1), PARP1 (poly(ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2),AGER (advanced glycosylation end product-specific receptor), IRS1(insulin receptor substrate 1), PTGS1 (prostaglandin-endoperoxidesynthase 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1(endothelin converting enzyme 1), F7 (coagulation factor VII (serumprothrombin conversion accelerator)), URN (interleukin 1 receptorantagonist), EPHX2 (epoxide hydrolase 2, cytoplasmic), IGFBP1(insulin-like growth factor binding protein 1), MAPK10(mitogen-activated protein kinase 10), FAS (Fas (TNF receptorsuperfamily, member 6)), ABCB1 (ATP-binding cassette, sub-family B(MDR/TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growthfactor binding protein 3), CD14 (CD14 molecule), PDE5A(phosphodiesterase 5A, cGMP-specific), AGTR2 (angiotensin II receptor,type 2), CD40 (CD40 molecule, TNF receptor superfamily member 5), LCAT(lecithin-cholesterol acyltransferase), CCR5 (chemokine (C-C motif)receptor 5), MMP1 (matrix metallopeptidase 1 (interstitialcollagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1), ADM(adrenomedullin), DYT10 (dystonia 10), STAT3 (signal transducer andactivator of transcription 3 (acute-phase response factor)), MMP3(matrix metallopeptidase 3 (stromelysin 1, progelatinase)), ELN(elastin), USF1 (upstream transcription factor 1), CFH (complementfactor H), HSPA4 (heat shock 70 kDa protein 4), MMP12 (matrixmetallopeptidase 12 (macrophage elastase)), MME (membranemetallo-endopeptidase), F2R (coagulation factor II (thrombin) receptor),SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1(adrenergic, beta-1-, receptor), CYBA (cytochrome b-245, alphapolypeptide), FGA (fibrinogen alpha chain), GGT1(gamma-glutamyltransferase 1), LIPG (lipase, endothelial), HIF1A(hypoxia inducible factor 1, alpha subunit (basic helix-loop-helixtranscription factor)), CXCR4 (chemokine (C-X-C motif) receptor 4), PROC(protein C (inactivator of coagulation factors Va and VIIIa)), SCARB1(scavenger receptor class B, member 1), CD79A (CD79a molecule,immunoglobulin-associated alpha), PLTP (phospholipid transfer protein),ADD1 (adducin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serumamyloid A1), KCNH2 (potassium voltage-gated channel, subfamily H(eag-related), member 2), DPP4 (dipeptidyl-peptidase 4), G6PD(glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptorA/guanylate cyclase A (atrionatriuretic peptide receptor A)), VTN(vitronectin), KIAA0101 (KIAA0101), FOS (FBJ murine osteosarcoma viraloncogene homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolylisomerase G (cyclophilin G)), IL1R1 (interleukin 1 receptor, type I), AR(androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A,polypeptide 1), SERPINA1 (serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 1), MTR(5-methyltetrahydrofolate-homocysteine methyltransferase), RBP4 (retinolbinding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A(cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)),FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptortype B), ITGA2 (integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2receptor)), CABIN1 (calcineurin binding protein 1), SHBG (sexhormone-binding globulin), HMGB1 (high-mobility group box 1), HSP90B2P(heat shock protein 90 kDa beta (Grp94), member 2 (pseudogene)), CYP3A4(cytochrome P450, family 3, subfamily A, polypeptide 4), GJA1 (gapjunction protein, alpha 1, 43 kDa), CAV1 (caveolin 1, caveolae protein,22 kDa), ESR2 (estrogen receptor 2 (ER beta)), LTA (lymphotoxin alpha(TNF superfamily, member 1)), GDF15 (growth differentiation factor 15),BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450,family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (betapolypeptide)), SP1 (Sp1 transcription factor), TGIF1 (TGFB-inducedfactor homeobox 1), SRC (v-src sarcoma (Schmidt-Ruppin A-2) viraloncogene homolog (avian)), EGF (epidermal growth factor(beta-urogastrone)), PIK3CG (phosphoinositide-3-kinase, catalytic, gammapolypeptide), HLA-A (major histocompatibility complex, class I, A),KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1),CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (cholinekinase alpha), BEST1 (bestrophin 1), APP (amyloid beta (A4) precursorprotein), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondinreceptor)), PRKAB1 (protein kinase, AMP-activated, beta 1 non-catalyticsubunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7family, member A1), CX3CR1 (chemokine (C-X3-C motif) receptor 1), TH(tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone1), TF (transferrin), HFE (hemochromatosis), IL17A (interleukin 17A),PTEN (phosphatase and tensin homolog), GSTM1 (glutathione S-transferasemu 1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1(coagulation factor XIII, A1 polypeptide), TTR (transthyretin), FABP4(fatty acid binding protein 4, adipocyte), PON3 (paraoxonase 3), APOC1(apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosisfactor receptor superfamily, member 1B), HTR2A (5-hydroxytryptamine(serotonin) receptor 2A), CSF3 (colony stimulating factor 3(granulocyte)), CYP2C9 (cytochrome P450, family 2, subfamily C,polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11,subfamily B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colonystimulating factor 2 (granulocyte-macrophage)), KDR (kinase insertdomain receptor (a type III receptor tyrosine kinase)), PLA2G2A(phospholipase A2, group IIA (platelets, synovial fluid)), B2M(beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA(ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2family (mitochondrial)), TCF7L2 (transcription factor 7-like 2 (T-cellspecific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclearfactor (erythroid-derived 2)-like 2), NOTCH1 (Notch homolog 1,translocation-associated (Drosophila)), UGT1A1 (UDPglucuronosyltransferase 1 family, polypeptide A1), IFNA1 (interferon,alpha 1), PPARD (peroxisome proliferator-activated receptor delta),SIRT1 (sirtuin (silent mating type information regulation 2 homolog) 1(S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1(luteinizing-releasing hormone)), PAPPA (pregnancy-associated plasmaprotein A, pappalysin 1), ARR3 (arrestin 3, retinal (X-arrestin)), NPPC(natriuretic peptide precursor C), AHSP (alpha hemoglobin stabilizingprotein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13),MTOR (mechanistic target of rapamycin (serine/threonine kinase)), ITGB2(integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)),GSTT1 (glutathione S-transferase theta 1), IL6ST (interleukin 6 signaltransducer (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase B2(plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrierfamily 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6(phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11(tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solutecarrier family 8 (sodium/calcium exchanger), member 1), F2RL1(coagulation factor II (thrombin) receptor-like 1), AKR1A1 (aldo-ketoreductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehydedehydrogenase 9 family, member A1), BGLAP (bone gamma-carboxyglutamate(gla) protein), MTTP (microsomal triglyceride transfer protein), MTRR(5-methyltetrahydrofolate-homocysteine methyltransferase reductase),SULT1A3 (sulfotransferase family, cytosolic, 1A, phenol-preferring,member 3), RAGE (renal tumor antigen), C4B (complement component 4B(Chido blood group), P2RY12 (purinergic receptor P2Y, G-protein coupled,12), RNLS (renalase, FAD-dependent amine oxidase), CREB1 (cAMPresponsive element binding protein 1), POMC (proopiomelanocortin), RAC1(ras-related C3 botulinum toxin substrate 1 (rho family, small GTPbinding protein Rac1)), LMNA (lamin NC), CD59 (CD59 molecule, complementregulatory protein), SCN5A (sodium channel, voltage-gated, type V, alphasubunit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide1), MIF (macrophage migration inhibitory factor(glycosylation-inhibiting factor)), MMP13 (matrix metallopeptidase 13(collagenase 3)), TIMP2 (TIMP metallopeptidase inhibitor 2), CYP19A1(cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2(cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22(protein tyrosine phosphatase, non-receptor type 22 (lymphoid)), MYH14(myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin(protein C) 2, soluble (opsonic defect)), SELPLG (selectin P ligand),AOC3 (amine oxidase, copper containing 3 (vascular adhesion protein 1)),CTSL1 (cathepsin L1), PCNA (proliferating cell nuclear antigen), IGF2(insulin-like growth factor 2 (somatomedin A)), ITGB1 (integrin, beta 1(fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2,MSK12)), CAST (calpastatin), CXCL12 (chemokine (C-X-C motif) ligand 12(stromal cell-derived factor 1)), IGHE (immunoglobulin heavy constantepsilon), KCNE1 (potassium voltage-gated channel, Isk-related family,member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen,type I, alpha 1), COL1A2 (collagen, type I, alpha 2), IL2RB (interleukin2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2(angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4(NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11(protein tyrosine phosphatase, non-receptor type 11), SLC2A1 (solutecarrier family 2 (facilitated glucose transporter), member 1), IL2RA(interleukin 2 receptor, alpha), CCL5 (chemokine (C-C motif) ligand 5),IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-likeapoptosis regulator), CALCA (calcitonin-related polypeptide alpha),EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (glutathioneS-transferase pi 1), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450,family 3, subfamily A, polypeptide 5), HSPG2 (heparan sulfateproteoglycan 2), CCL3 (chemokine (C-C motif) ligand 3), MYD88 (myeloiddifferentiation primary response gene (88)), VIP (vasoactive intestinalpeptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenergic, beta,receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member2), MMP8 (matrix metallopeptidase 8 (neutrophil collagenase)), NPR2(natriuretic peptide receptor B/guanylate cyclase B (atrionatriureticpeptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS(glutamyl-prolyl-tRNA synthetase), PPARGC1A (peroxisomeproliferator-activated receptor gamma, coactivator 1 alpha), F12(coagulation factor XII (Hageman factor)), PECAM1 (platelet/endothelialcell adhesion molecule), CCL4 (chemokine (C-C motif) ligand 4), SERPINA3(serpin peptidase inhibitor, clade A (alpha-1 antiproteinase,antitrypsin), member 3), CASR (calcium-sensing receptor), GJA5 (gapjunction protein, alpha 5, 40 kDa), FABP2 (fatty acid binding protein 2,intestinal), TTF2 (transcription termination factor, RNA polymerase II),PROS1 (protein S (alpha)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan,beta (43 kDa dystrophin-associated glycoprotein)), YME1L1 (YME1-like 1(S. cerevisiae)), CAMP (cathelicidin antimicrobial peptide), ZC3H12A(zinc finger CCCH-type containing 12A), AKR1B1 (aldo-keto reductasefamily 1, member B1 (aldose reductase)), DES (desmin), MMP7 (matrixmetallopeptidase 7 (matrilysin, uterine)), AHR (aryl hydrocarbonreceptor), CSF1 (colony stimulating factor 1 (macrophage)), HDAC9(histone deacetylase 9), CTGF (connective tissue growth factor), KCNMA1(potassium large conductance calcium-activated channel, subfamily M,alpha member 1), UGT1A (UDP glucuronosyltransferase 1 family,polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT(catechol-.beta.-methyltransferase), S100B (S100 calcium binding proteinB), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin15), DRD4 (dopamine receptor D4), CAMK2G (calcium/calmodulin-dependentprotein kinase II gamma), SLC22A2 (solute carrier family 22 (organiccation transporter), member 2), CCL11 (chemokine (C-C motif) ligand 11),PGF (B321 placental growth factor), THPO (thrombopoietin), GP6(glycoprotein VI (platelet)), TACR1 (tachykinin receptor 1), NTS(neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1(potassium voltage-gated channel, Shal-related subfamily, member 1),LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1(platelet)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol dehydrogenase 1C(class I), gamma polypeptide), ALOX12 (arachidonate 12-lipoxygenase),AHSG (alpha-2-HS-glycoprotein), BHMT (betaine-homocysteinemethyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa),SLC25A4 (solute carrier family 25 (mitochondrial carrier; adeninenucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP(arachidonate 5-lipoxygenase-activating protein), NUMA1 (nuclear mitoticapparatus protein 1), CYP27B1 (cytochrome P450, family 27, subfamily B,polypeptide 1), CYSLTR2 (cysteinyl leukotriene receptor 2), SOD3(superoxide dismutase 3, extracellular), LTC4S (leukotriene C4synthase), UCN (urocortin), GHRL (ghrelin/obestatin prepropeptide),APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4,member A), KBTBD10 (kelch repeat and BTB (POZ) domain containing 10),TNC (tenascin C), TYMS (thymidylate synthetase), SHCl (SHC (Src homology2 domain containing) transforming protein 1), LRP1 (low densitylipoprotein receptor-related protein 1), SOCS3 (suppressor of cytokinesignaling 3), ADH1B (alcohol dehydrogenase 1B (class I), betapolypeptide), KLK3 (kallikrein-related peptidase 3), HSD11B1(hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxidereductase complex, subunit 1), SERPINB2 (serpin peptidase inhibitor,clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring fingerprotein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, alpha M(complement component 3 receptor 3 subunit)), PITX2 (paired-likehomeodomain 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (Fcfragment of IgG, low affinity 111a, receptor (CD16a)), LEPR (leptinreceptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2(glutamic-oxaloacetic transaminase 2, mitochondrial (aspartateaminotransferase 2)), HRH1 (histamine receptor H1), NR112 (nuclearreceptor subfamily 1, group I, member 2), CRH (corticotropin releasinghormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1(voltage-dependent anion channel 1), HPSE (heparanase), SFTPD(surfactant protein D), TAP2 (transporter 2, ATP-binding cassette,sub-family B (MDR/TAP)), RNF123 (ring finger protein 123), PTK2B (PTK2Bprotein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase,receptor, type 2), IL6R (interleukin 6 receptor), ACHE(acetylcholinesterase (Yt blood group)), GLP1R (glucagon-like peptide 1receptor), GHR (growth hormone receptor), GSR (glutathione reductase),NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptorsubfamily 5, group A, member 1), GJB2 (gap junction protein, beta 2, 26kDa), SLC9A1 (solute carrier family 9 (sodium/hydrogen exchanger),member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertasesubtilisin/kexin type 9), FCGR2A (Fc fragment of IgG, low affinity IIa,receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, clade F (alpha-2antiplasmin, pigment epithelium derived factor), member 1), EDN3(endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growtharrest-specific 6), SMPD1 (sphingomyelin phosphodiesterase 1, acidlysosomal), UCP2 (uncoupling protein 2 (mitochondrial, proton carrier)),TFAP2A (transcription factor AP-2 alpha (activating enhancer bindingprotein 2 alpha)), C4BPA (complement component 4 binding protein,alpha), SERPINF2 (serpin peptidase inhibitor, clade F (alpha-2antiplasmin, pigment epithelium derived factor), member 2), TYMP(thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Reganisozyme)), CXCR2 (chemokine (C-X-C motif) receptor 2), SLC39A3 (solutecarrier family 39 (zinc transporter), member 3), ABCG2 (ATP-bindingcassette, sub-family G (WHITE), member 2), ADA (adenosine deaminase),JAK3 (Janus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN(fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), F11(coagulation factor XI), ATP7A (ATPase, Cu++ transporting, alphapolypeptide), CR1 (complement component (3b/4b) receptor 1 (Knops bloodgroup)), GFAP (glial fibrillary acidic protein), ROCK1 (Rho-associated,coiled-coil containing protein kinase 1), MECP2 (methyl CpG bindingprotein 2 (Rett syndrome)), MYLK (myosin light chain kinase), BCHE(butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5(peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome,RecQ helicase-like), CXCR3 (chemokine (C-X-C motif) receptor 3), CD81(CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, gamma 2),MAP3K5 (mitogen-activated protein kinase kinase kinase 5), CHGA(chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloidpolypeptide), RHO (rhodopsin), ENPP1 (ectonucleotidepyrophosphatase/phosphodiesterase 1), PTHLH (parathyroid hormone-likehormone), NRG1 (neuregulin 1), VEGFC (vascular endothelial growth factorC), ENPEP (glutamyl aminopeptidase (aminopeptidase A)), CEBPB(CCAAT/enhancer binding protein (C/EBP), beta), NAGLU(N-acetylglucosaminidase, alpha-), F2RL3 (coagulation factor II(thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif) ligand 1),BDKRB1 (bradykinin receptor B1), ADAMTS13 (ADAM metallopeptidase withthrombospondin type 1 motif, 13), ELANE (elastase, neutrophilexpressed), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2),CISH (cytokine inducible SH2-containing protein), GAST (gastrin), MYOC(myocilin, trabecular meshwork inducible glucocorticoid response),ATP1A2 (ATPase, Na+/K+ transporting, alpha 2 polypeptide), NF1(neurofibromin 1), GJB1 (gap junction protein, beta 1, 32 kDa), MEF2A(myocyte enhancer factor 2A), VCL (vinculin), BMPR2 (bone morphogeneticprotein receptor, type II (serine/threonine kinase)), TUBB (tubulin,beta), CDCl42 (cell division cycle 42 (GTP binding protein, 25 kDa)),KRT18 (keratin 18), HSF1 (heat shock transcription factor 1), MYB (v-mybmyeloblastosis viral oncogene homolog (avian)), PRKAA2 (protein kinase,AMP-activated, alpha 2 catalytic subunit), ROCK2 (Rho-associated,coiled-coil containing protein kinase 2), TFPI (tissue factor pathwayinhibitor (lipoprotein-associated coagulation inhibitor)), PRKG1(protein kinase, cGMP-dependent, type I), BMP2 (bone morphogeneticprotein 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CTH(cystathionase (cystathionine gamma-lyase)), CTSS (cathepsin S), VAV2(vav 2 guanine nucleotide exchange factor), NPY2R (neuropeptide Yreceptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase alpha 1),PPIA (peptidylprolyl isomerase A (cyclophilin A)), APOH (apolipoproteinH (beta-2-glycoprotein I)), S100A8 (S100 calcium binding protein A8),IL11 (interleukin 11), ALOX15 (arachidonate 15-lipoxygenase), FBLN1(fibulin 1), NR1H3 (nuclear receptor subfamily 1, group H, member 3),SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastricinhibitory polypeptide), CHGB (chromogranin B (secretogranin 1)), PRKCB(protein kinase C, beta), SRD5A1 (steroid-5-alpha-reductase, alphapolypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1)),HSD11B2 (hydroxysteroid (11-beta) dehydrogenase 2), CALCRL (calcitoninreceptor-like), GALNT2 (UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase 2 (GalNAc-T2)), ANGPTL4(angiopoietin-like 4), KCNN4 (potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4), PIK3C2A(phosphoinositide-3-kinase, class 2, alpha polypeptide), HBEGF(heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450,family 7, subfamily A, polypeptide 1), HLA-DRB5 (majorhistocompatibility complex, class II, DR beta 5), BNIP3 (BCL2/adenovirusE1B 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4)regulator), S100A12 (S100 calcium binding protein A12), PADI4 (peptidylarginine deiminase, type IV), HSPA14 (heat shock 70 kDa protein 14),CXCR1 (chemokine (C-X-C motif) receptor 1), H19 (H19, imprintedmaternally expressed transcript (non-protein coding)), KRTAP19-3(keratin associated protein 19-3), IDDM2 (insulin-dependent diabetesmellitus 2), RAC2 (ras-related C3 botulinum toxin substrate 2 (rhofamily, small GTP binding protein Rac2)), RYR1 (ryanodine receptor 1(skeletal)), CLOCK (clock homolog (mouse)), NGFR (nerve growth factorreceptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase(dopamine beta-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic,alpha 4), CACNA1C (calcium channel, voltage-dependent, L type, alpha 1Csubunit), PRKAG2 (protein kinase, AMP-activated, gamma 2 non-catalyticsubunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H,member 2), TEK (TEK tyrosine kinase, endothelial), VEGFB (vascularendothelial growth factor B), MEF2C (myocyte enhancer factor 2C),MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2),TNFRSF11A (tumor necrosis factor receptor superfamily, member 11a, NFKBactivator), HSPA9 (heat shock 70 kDa protein 9 (mortalin)), CYSLTR1(cysteinyl leukotriene receptor 1), MAT1A (methionineadenosyltransferase I, alpha), OPRL1 (opiate receptor-like 1), IMPA1(inositol(myo)-1(or 4)-monophosphatase 1), CLCN2 (chloride channel 2),DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome (prosome,macropain) subunit, alpha type, 6), PSMB8 (proteasome (prosome,macropain) subunit, beta type, 8 (large multifunctional peptidase 7)),CHI3L1 (chitinase 3-like 1 (cartilage glycoprotein-39)), ALDH1B1(aldehyde dehydrogenase 1 family, member B1), PARP2 (poly (ADP-ribose)polymerase 2), STAR (steroidogenic acute regulatory protein), LBP(lipopolysaccharide binding protein), ABCC6 (ATP-binding cassette,sub-family C(CFTR/MRP), member 6), RGS2 (regulator of G-proteinsignaling 2, 24 kDa), EFNB2 (ephrin-B2), GJB6 (gap junction protein,beta 6, 30 kDa), APOA2 (apolipoprotein A-II), AMPD1 (adenosinemonophosphate deaminase 1), DYSF (dysferlin, limb girdle musculardystrophy 2B (autosomal recessive)), FDFT1 (farnesyl-diphosphatefarnesyltransferase 1), EDN2 (endothelin 2), CCR6 (chemokine (C-C motif)receptor 6), GJB3 (gap junction protein, beta 3, 31 kDa), IL1RL1(interleukin 1 receptor-like 1), ENTPD1 (ectonucleoside triphosphatediphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (cadherin,EGF LAG seven-pass G-type receptor 2 (flamingo homolog, Drosophila)),F11R (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor(GEF) 3), HYAL1 (hyaluronoglucosaminidase 1), ZNF259 (zinc fingerprotein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)), ATF6(activating transcription factor 6), KHK (ketohexokinase(fructokinase)), SAT1 (spermidine/spermine N1-acetyltransferase 1), GGH(gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamylhydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solutecarrier family 4, sodium bicarbonate cotransporter, member 4), PDE2A(phosphodiesterase 2A, cGMP-stimulated), PDE3B (phosphodiesterase 3B,cGMP-inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty aciddesaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxininteracting protein), LIMS1 (LIM and senescent cell antigen-like domains1), RHOB (ras homolog gene family, member B), LY96 (lymphocyte antigen96), FOXO1 (forkhead box O1), PNPLA2 (patatin-like phospholipase domaincontaining 2), TRH (thyrotropin-releasing hormone), GJC1 (gap junctionprotein, gamma 1, 45 kDa), SLC17A5 (solute carrier family 17(anion/sugar transporter), member 5), FTO (fat mass and obesityassociated), GJD2 (gap junction protein, delta 2, 36 kDa), PSRC1(proline/serine-rich coiled-coil 1), CASP12 (caspase 12(gene/pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK(PX domain containing serine/threonine kinase), IL33 (interleukin 33),TRIB1 (tribbles homolog 1 (Drosophila)), PBX4 (pre-B-cell leukemiahomeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1),15-Sep (15 kDa selenoprotein), CILP2 (cartilage intermediate layerprotein 2), TERC (telomerase RNA component), GGT2(gamma-glutamyltransferase 2), MT-CO1 (mitochondrially encodedcytochrome c oxidase I), and UOX (urate oxidase, pseudogene). Any ofthese sequences, may be a target for the CRISPR-Cas system, e.g., toaddress mutation.

In an additional embodiment, the chromosomal sequence may further beselected from Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE(Apolipoprotein E), Apo B-100 (Apolipoprotein B-100), ApoA(Apolipoprotein(a)), ApoA1 (Apolipoprotein A1), CBS (CystathioneB-synthase), Glycoprotein IIb/IIb, MTHRF (5,10-methylenetetrahydrofolatereductase (NADPH), and combinations thereof. In one iteration, thechromosomal sequences and proteins encoded by chromosomal sequencesinvolved in cardiovascular disease may be chosen from Cacna1C, Sod1,Pten, Ppar(alpha), Apo E, Leptin, and combinations thereof as target(s)for the CRISPR-Cas system.

Treating Diseases of the Liver and Kidney

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. Cpf1 effector protein systems, to the liverand/or kidney. Delivery strategies to induce cellular uptake of thetherapeutic nucleic acid include physical force or vector systems suchas viral-, lipid- or complex-based delivery, or nanocarriers. From theinitial applications with less possible clinical relevance, when nucleicacids were addressed to renal cells with hydrodynamic high pressureinjection systemically, a wide range of gene therapeutic viral andnon-viral carriers have been applied already to targetposttranscriptional events in different animal kidney disease models invivo (Csaba Révész and Peter Hamar (2011). Delivery Methods to TargetRNAs in the Kidney, Gene Therapy Applications, Prof. Chunsheng Kang(Ed.), ISBN: 978-953-307-541-9, InTech, Available from:http://www.intechopen.com/books/gene-thorapy-applications/delivery-methods-to-target-rnas-inthe-kidney).Delivery methods to the kidney may include those in Yuan et al. (Am JPhysiol Renal Physiol 295: F605-F617, 2008) investigated whether in vivodelivery of small interfering RNAs (siRNAs) targeting the12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolismcan ameliorate renal injury and diabetic nephropathy (DN) in astreptozotocininjected mouse model of type 1 diabetes. To achievegreater in vivo access and siRNA expression in the kidney, Yuan et al.used double-stranded 12/15-LO siRNA oligonucleotides conjugated withcholesterol. About 400 μg of siRNA was injected subcutaneously intomice. The method of Yuang et al. may be applied to the CRISPR Cas systemof the present invention contemplating a 1-2 g subcutaneous injection ofCRISPR Cas conjugated with cholesterol to a human for delivery to thekidneys.

Molitoris et al. (J Am Soc Nephrol 20: 1754-1764, 2009) exploitedproximal tubule cells (PTCs), as the site of oligonucleotidereabsorption within the kidney to test the efficacy of siRNA targeted top53, a pivotal protein in the apoptotic pathway, to prevent kidneyinjury. Naked synthetic siRNA to p53 injected intravenously 4 h afterischemic injury maximally protected both PTCs and kidney function.Molitoris et al.'s data indicates that rapid delivery of siRNA toproximal tubule cells follows intravenous administration. Fordose-response analysis, rats were injected with doses of siP53, 0.33; 1,3, or 5 mg/kg, given at the same four time points, resulting incumulative doses of 1.32; 4, 12, and 20 mg/kg, respectively. All siRNAdoses tested produced a SCr reducing effect on day one with higher dosesbeing effective over approximately five days compared with PBS-treatedischemic control rats. The 12 and 20 mg/kg cumulative doses provided thebest protective effect. The method of Molitoris et al. may be applied tothe nucleic acid-targeting system of the present invention contemplating12 and 20 mg/kg cumulative doses to a human for delivery to the kidneys.

Thompson et al. (Nucleic Acid Therapeutics, Volume 22, Number 4, 2012)reports the toxicological and pharmacokinetic properties of thesynthetic, small interfering RNA I5NP following intravenousadministration in rodents and nonhuman primates. I5NP is designed to actvia the RNA interference (RNAi) pathway to temporarily inhibitexpression of the pro-apoptotic protein p53 and is being developed toprotect cells from acute ischemia/reperfusion injuries such as acutekidney injury that can occur during major cardiac surgery and delayedgraft function that can occur following renal transplantation. Doses of800 mg/kg I5NP in rodents, and 1,000 mg/kg I5NP in nonhuman primates,were required to elicit adverse effects, which in the monkey wereisolated to direct effects on the blood that included a sub-clinicalactivation of complement and slightly increased clotting times. In therat, no additional adverse effects were observed with a rat analogue ofI5NP, indicating that the effects likely represent class effects ofsynthetic RNA duplexes rather than toxicity related to the intendedpharmacologic activity of I5NP. Taken together, these data supportclinical testing of intravenous administration of I5NP for thepreservation of renal function following acute ischemia/reperfusioninjury. The no observed adverse effect level (NOAEL) in the monkey was500 mg/kg. No effects on cardiovascular, respiratory, and neurologicparameters were observed in monkeys following i.v. administration atdose levels up to 25 mg/kg. Therefore, a similar dosage may becontemplated for intravenous administration of CRISPR Cas to the kidneysof a human.

Shimizu et al. (J Am Soc Nephrol 21: 622-633, 2010) developed a systemto target delivery of siRNAs to glomeruli via poly(ethyleneglycol)-poly(L-lysine)-based vehicles. The siRNA/nanocarrier complex wasapproximately 10 to 20 nm in diameter, a size that would allow it tomove across the fenestrated endothelium to access to the mesangium.After intraperitoneal injection of fluorescence-labeledsiRNA/nanocarrier complexes, Shimizu et al. detected siRNAs in the bloodcirculation for a prolonged time. Repeated intraperitonealadministration of a mitogen-activated protein kinase 1 (MAPK1)siRNA/nanocarrier complex suppressed glomerular MAPK1 mRNA and proteinexpression in a mouse model of glomerulonephritis. For the investigationof siRNA accumulation, Cy5-labeled siRNAs complexed with PICnanocarriers (0.5 ml, 5 nmol of siRNA content), naked Cy5-labeled siRNAs(0.5 ml, 5 nmol), or Cy5-labeled siRNAs encapsulated in HVJ-E (0.5 ml, 5nmol of siRNA content) were administrated to BALBc mice. The method ofShimizu et al. may be applied to the nucleic acid-targeting system ofthe present invention contemplating a dose of about of 10-20 μmol CRISPRCas complexed with nanocarriers in about 1-2 liters to a human forintraperitoneal administration and delivery to the kidneys.

Delivery methods to the kidney are summarized as follows:

Delivery Target Functional method Carrier RNA Disease Model assaysAuthor Hydrodynamic/ TransIT In p85α Acute Ischemia- Uptake, Larson etal., Lipid Vivo Gene renal reperfusion biodistribution Surgery, Deliveryinjury (August 2007), System, Vol. 142, No. 2, DOTAP pp. (262-269)Hydrodynamic/ Lipofectamine Fas Acute Ischemia- Blood urea nitrogen,Hamar et al., Lipid 2000 renal reperfusion Fas Immunohistochemistry,Proc Natl Acad injury apoptosis, histological Sci, (October 2004),scoring Vol. 101, No. 41, pp. (14883-14888) Hydrodynamic n.a. ApoptosisAcute Ischemia- n.a. Zheng et al., cascade renal reperfusion Am JPathol, elements injury (October 2008), Vol. 173, No. 4, pp. (973-980)Hydrodynamic n.a. Nuclear Acute Ischemia- n.a. Feng et al., factor renalreperfusion Transplantation, kappa-b injury (May 2009), (NFkB) Vol. 87,No. 9, pp. (1283-1289) Hydrodynamic/ Lipofectamine Apoptosis AcuteIschemia- Apoptosis, oxidative Xie & Guo, Am Soc Viral 2000 antagonizingrenal reperfusion stress, caspase Nephrol, transcription injuryactivation, membrane (December 2006), factor (AATF) lipid peroxidationVol. 17, No. 12, pp. (3336-3346) Hydrodynmic pBAsi mU6 Gremlin DiabeticStreptozotozin - Proteinuria, serum Q. Zhang et al., Neo/TransIT-nephropathy induced creatinine, glomerular PloS ONE, (July EEHydrodynamic diabetes and tubular diameter, 2010), Vol. 5, DeliverySystem collagen type IV/BMP7 No. 7, e11709, expression pp. (1-13) Viral/pSUPER TGF-β Interstitial Unilateral α-SMA expression, Kushibikia etal., J Lipid vector/ type II renal urethral collagen content, ControlledRelease, Lipofectamine receptor fibrosis obstruction (July 2005), Vol.105, No. 3, pp. (318-331) Viral Adeno- Mineral Hypertension Cold- bloodpressure, Wang et al., associated corticoid caused renal induced serumalbumin, Gene Therapy, virus-2 receptor damage hypertension serum urea(July 2006), nitrogen, serum Vol. 13, No. creatinine, kidney 14, pp.(1097-1103) weight, urinary sodium Hydrodynamic/ pU6 vector Luciferasen.a. n.a. uptake Kobayashi et Viral al., Journal of Pharmacology andExperimental Therapeutics, (February 2004), Vol. 308, No. 2, pp.(688-693) Lipid Lipoproteins, apoB1, n.a. n.a. Uptake, binding Wolfrumet albumin apoM affinity to al., Nature lipoproteins Biotechnology, andalbumin (September 2007), Vol. 25, No. 10, pp. (1149-1157) LipidLipofectamine2000 p53 Acute Ischemic and Histological Molitoris et renalcisplatin- scoring, al., J Am Soc injury induced apoptosis Nephrol,acute injury (August 2009), Vol. 20, No. 8, pp. (1754-1764) LipidDOTAP/DOPE, COX-2 Breast MDA-MB-231 Cell viability, Mikhaylova etDOTAP/DOPE/ adenocarcinoma breast cancer uptake al., Cancer Gene DOPE-xenograft- Therapy, PEG2000 bearing (March 2011), mouse Vol. 16, No. 3,pp. (217-226) Lipid Cholesterol 12/15- Diabetic Streptozotocin -Albuminuria, urinary Yuan et al., lipoxygenase nephropathy inducedcreatinine, histology, Am J Physiol diabetes type I and IV collagen,Renal Physiol, TGF-β, fibronectin, (June 2008), plasminogen activatorVol. 295, pp. inhibitor 1 (F605-F617) Lipid Lipofectamine MitochondrialDiabetic Streptozotocin - Cell proliferation Y. Zhang et 2000 membranenephropathy induced and apoptosis, al., J Am 44 (TIM44) diabeteshistology, ROS, Soc Nephrol, mitochondrial import of (April 2006), Vol.Mn-SOD and glutathione 17, No. 4, pp. peroxidase, cellular (1090-1101)membrane polarization Hydrodynamic/ Proteoliposome RLIP76 Renal Caki-2uptake Singhal et al., Lipid carcinoma kidney cancer Cancer Res,xenograft- (May 2009), bearing mouse Vol. 69, No. 10, pp. (4244- 4251)Polymer PEGylated PEI Luciferase n.a. n.a. Uptake, Malek et al., pGL3biodistribution, Toxicology erythrocyte and Applied aggregationPharmacology, (April 2009), Vol. 236, No. 1, pp. (97-108) PolymerPEGylated MAPK1 Lupus Glomerulonephritis Proteinuria, Shimizu et al.,poly-L-lysine glomerulonephritis glomerulosclerosis, J Am Soc TGF- β,Nephrology, fibronectin, (April 2010), plasminogen Vol. 21, No. 4,activator pp. (622-633) inhibitor 1 Polymer/ Hyaluronic VEGF KidneyB16F1 Biodistribution, Jiang et al., Nano particle acid/Quantum cancer/melanoma citotoxicity, Molecular dot/PEI melanoma tumor- tumor volume,Pharmaceutics, bearing endocytosis (May-June 2009), mouse Vol. 6, No. 3,pp. (727-737) Polymer/ PEGylated GAPDH n.a. n.a. cell viability, Cao etal, J Nano particle polycaprolactone uptake Controlled nanofiberRelease, (June 2010), Vol. 144, No. 2, pp. (203-212) Aptamer SpiegelmerCC Glomerulosclerosis Uninephrecto- urinary albumin, Ninichuk etmNOX-E36 chemokine mized mouse urinary creatinine, al., Am J Pathol,ligand 2 histopathology, (March 2008), glomerular Vol. 172, filtrationrate, No. 3, macrophage count, pp. (628-637) serum Ccl2, Mac- 2+, ki-67+Aptamer Aptamer NOX- vasopressin Congestive n.a. Binding affinity to D-Purschke et F37 (AVP) heart failure AVP, Inhibition of al., Proc NatlAVP Signaling, Urine Acad Sci, osmolality and (March 2006), sodiumconcentration, Vol. 103, No. 13, pp. (5173-5178)

Targeting the Liver or Liver Cells

Targeting liver cells is provided. This may be in vitro or in vivo.Hepatocytes are preferred. Delivery of the CRISPR protein, such as Cpf1herein may be via viral vectors, especially AAV (and in particularAAV2/6) vectors. These may be administered by intravenous injection.

A preferred target for liver, whether in vitro or in vivo, is thealbumin gene. This is a so-called ‘safe harbor” as albumin is expressedat very high levels and so some reduction in the production of albuminfollowing successful gene editing is tolerated. It is also preferred asthe high levels of expression seen from the albumin promoter/enhancerallows for useful levels of correct or transgene production (from theinserted donor template) to be achieved even if only a small fraction ofhepatocytes are edited.

Intron 1 of albumin has been shown by Wechsler et al. (reported at the57th Annual Meeting and Exposition of the American Society ofHematology—abstract available online athttp://ash.confex.com/ash/2015/webprogram/Paper86495.html and presentedon 6th December 2015) to be a suitable target site. Their work used ZnFingers to cut the DNA at this target site, and suitable guide sequencescan be generated to guide cleavage at the same site by a CRISPR protein.

The use of targets within highly-expressed genes (genes with highlyactive enhancers/promoters) such as albumin may also allow apromoterless donor template to be used, as reported by Wechsler et al.and this is also broadly applicable outside liver targeting. Otherexamples of highly-expressed genes are known.

Other Disease of the Liver

In particular embodiments, the CRISPR proteins of the present inventionare used in the treatment of liver disorders such as transthyretinamyloidosis (ATTR), alpha-1 antitrypsin deficiency and otherhepatic-based inborn errors of metabolism. FAP is caused by a mutationin the gene that encodes transthyretin (TTR). While it is an autosomaldominant disease, not al carriers develop the disease. There are over100 mutations in the TTR gene known to be associated with the disease.Examples of common mutations include V30M. The principle of treatment ofTTR based on gene silencing has been demonstrated by studies with iRNA(Ueda et al. 2014 Transl Neurogener. 3:19). Wilson's Disease (WD) iscaused by mutations in the gene encoding ATP7B, which is foundexclusively in the hepatocyte. There are over 500 mutations associatedwith WD, with increased prevalence in specific regions such as EastAsia. Other examples are A1ATD (an autosomal recessive disease caused bymutations in the SERPINA1 gene) and PKU (an autosomal recessive diseasecaused by mutations in the phenylalanine hydroxylase (PAH) gene).

Liver-Associated Blood Disorders, especially Hemophilia and inparticular Hemophilia B

Successful gene editing of hepatocytes has been achieved in mice (bothin vitro and in vivo) and in non-human primates (in vivo), showing thattreatment of blood disorders through gene editing/genome engineering inhepatocytes is feasible. In particular, expression of the human F9 (hF9)gene in hepatocytes has been shown in non-human primates indicating atreatment for Hemophillia B in humans.

Wechsler et al. reported at the 57th Annual Meeting and Exposition ofthe American Society of Hematology (abstract presented 6 Dec. 2015 andavailable online athttps://ash.confex.com/ash/2015/webprogram/Paper86495.html) that theyhas successfully expressed human F9 (hF9) from hepatocytes in non-humanprimates through in vivo gene editing. This was achieved using 1) twozinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus,and 2) a human F9 donor template construct. The ZFNs and donor templatewere encoded on separate hepatotropic adeno-associated virus serotype2/6 (AAV2/6) vectors injected intravenously, resulting in targetedinsertion of a corrected copy of the hF9 gene into the albumin locus ina proportion of liver hepatocytes.

The albumin locus was selected as a “safe harbor” as production of thismost abundant plasma protein exceeds 10 g/day, and moderate reductionsin those levels are well-tolerated. Genome edited hepatocytes producednormal hFIX (hF9) in therapeutic quantities, rather than albumin, drivenby the highly active albumin enhancer/promoter. Targeted integration ofthe hF9 transgene at the albumin locus and splicing of this gene intothe albumin transcript was shown.

Mice studies: C57BL/6 mice were administered vehicle (n=20) or AAV2/6vectors (n=25) encoding mouse surrogate reagents at 1.0×1013 vectorgenome (vg)/kg via tail vein injection. ELISA analysis of plasma hFIX inthe treated mice showed peak levels of 50-1053 ng/mL that were sustainedfor the duration of the 6-month study. Analysis of FIX activity frommouse plasma confirmed bioactivity commensurate with expression levels.

Non-human primate (NHP) studies: a single intravenous co-infusion ofAAV2/6 vectors encoding the NHP targeted albumin-specific ZFNs and ahuman F9 donor at 1.2×1013 vg/kg (n=5/group) resulted in >50 ng/mL (>1%of normal) in this large animal model. The use of higher AAV2/6 doses(up to 1.5×1014 vg/kg) yielded plasma hFIX levels up to 1000 ng/ml (or20% of normal) in several animals and up to 2000 ng/ml (or 50% ofnormal) in a single animal, for the duration of the study (3 months).

The treatment was well tolerated in mice and NHPs, with no significanttoxicological findings related to AAV2/6 ZFN+donor treatment in eitherspecies at therapeutic doses. Sangamo (CA, USA) has since applied to theFDA, and been granted, permission to conduct the world's first humanclinical trial for an in vivo genome editing application. This followson the back of the EMEA's approval of the Glybera gene therapy treatmentof lipoprotein lipase deficiency.

Accordingly, it is preferred, in some embodiments, that any or all ofthe following are used:

-   -   AAV (especially AAV2/6) vectors, preferably administered by        intravenous injection;    -   Albumin as target for gene editing/insertion of        transgene/template—especially at intron 1 of albumin;    -   human F9 donor template; and/or    -   a promoterless donor template.

Hemophilia B

Accordingly, in some embodiments, it is preferred that the presentinvention is used to treat Hemophilia B. As such it is preferred that atemplate is provided and that this is the human F9 gene. It will beappreciated that the hF9 template comprises the wt or ‘correct’ versionof hF9 so that the treatment is effective.

In an alternative embodiment, the hemophilia B version of F9 may bedelivered so as to create a model organism, cell or cell line (forexample a murine or non-human primate model organism, cell or cellline), the model organism, cell or cell line having or carrying theHemophilia B phenotype, i.e. an inability to produce wt F9.

Hemophilia A

In some embodiments, the F9 (factor IX) gene may be replaced by the F8(factor VIII) gene described above, leading to treatment of Hemophilia A(through provision of a correct F8 gene) and/or creation of a HemophiliaA model organism, cell or cell line (through provision of an incorrect,Hemophilia A version of the F8 gene).

Hemophilia C

In some embodiments, the F9 (factor IX) gene may be replaced by the F11(factor XI) gene described above, leading to treatment of Hemophilia C(through provision of a correct F11 gene) and/or creation of aHemophilia C model organism, cell or cell line (through provision of anincorrect, Hemophilia C version of the F11 gene).

Treating Epithelial and Lung Diseases

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. Cpf1 effector protein systems, to one or bothlungs.

Although AAV-2-based vectors were originally proposed for CFTR deliveryto CF airways, other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9exhibit improved gene transfer efficiency in a variety of models of thelung epithelium (see, e.g., Li et al., Molecular Therapy, vol. 17 no.12, 2067-277 December 2009). AAV-1 was demonstrated to be ˜100-fold moreefficient than AAV-2 and AAV-5 at transducing human airway epithelialcells in vitro, 5 although AAV-1 transduced murine tracheal airwayepithelia in vivo with an efficiency equal to that of AAV-5. Otherstudies have shown that AAV-5 is 50-fold more efficient than AAV-2 atgene delivery to human airway epithelium (HAE) in vitro andsignificantly more efficient in the mouse lung airway epithelium invivo. AAV-6 has also been shown to be more efficient than AAV-2 in humanairway epithelial cells in vitro and murine airways in vivo.8 The morerecent isolate, AAV-9, was shown to display greater gene transferefficiency than AAV-5 in murine nasal and alveolar epithelia in vivowith gene expression detected for over 9 months suggesting AAV mayenable long-term gene expression in vivo, a desirable property for aCFTR gene delivery vector. Furthermore, it was demonstrated that AAV-9could be readministered to the murine lung with no loss of CFTRexpression and minimal immune consequences. CF and non-CF HAE culturesmay be inoculated on the apical surface with 100 μl of AAV vectors forhours (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-277December 2009). The MOI may vary from 1×10³ to 4×10⁵ vectorgenomes/cell, depending on virus concentration and purposes of theexperiments. The above cited vectors are contemplated for the deliveryand/or administration of the invention.

Zamora et al. (Am J Respir Crit Care Med Vol 183. pp 531-538, 2011)reported an example of the application of an RNA interferencetherapeutic to the treatment of human infectious disease and also arandomized trial of an antiviral drug in respiratory syncytial virus(RSV)-infected lung transplant recipients. Zamora et al. performed arandomized, double-blind, placebocontrolled trial in LTX recipients withRSV respiratory tract infection. Patients were permitted to receivestandard of care for RSV. Aerosolized ALN-RSV01 (0.6 mg/kg) or placebowas administered daily for 3 days. This study demonstrates that an RNAitherapeutic targeting RSV can be safely administered to LTX recipientswith RSV infection. Three daily doses of ALN-RSV01 did not result in anyexacerbation of respiratory tract symptoms or impairment of lungfunction and did not exhibit any systemic proinflammatory effects, suchas induction of cytokines or CRP. Pharmacokinetics showed only low,transient systemic exposure after inhalation, consistent withpreclinical animal data showing that ALN-RSV01, administeredintravenously or by inhalation, is rapidly cleared from the circulationthrough exonucleasemediated digestion and renal excretion. The method ofZamora et al. may be applied to the nucleic acid-targeting system of thepresent invention and an aerosolized CRISPR Cas, for example with adosage of 0.6 mg/kg, may be contemplated for the present invention.

Subjects treated for a lung disease may for example receivepharmaceutically effective amount of aerosolized AAV vector system perlung endobronchially delivered while spontaneously breathing. As such,aerosolized delivery is preferred for AAV delivery in general. Anadenovirus or an AAV particle may be used for delivery. Suitable geneconstructs, each operably linked to one or more regulatory sequences,may be cloned into the delivery vector. In this instance, the followingconstructs are provided as examples: Cbh or EF1a promoter for Cas(Cpf1), U6 or H1 promoter for guide RNA): A preferred arrangement is touse a CFTRdelta508 targeting guide, a repair template for deltaF508mutation and a codon optimized Cpf1 enzyme, with optionally one or morenuclear localization signal or sequence(s) (NLS(s)), e.g., two (2) NLSs.Constructs without NLS are also envisaged.

Treating Diseases of the Muscular System

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. Cpf1 effector protein systems, to muscle(s).

Bortolanza et al. (Molecular Therapy vol. 19 no. 11, 2055-264 November2011) shows that systemic delivery of RNA interference expressioncassettes in the FRG1 mouse, after the onset of facioscapulohumeralmuscular dystrophy (FSHD), led to a dose-dependent long-term FRG1knockdown without signs of toxicity. Bortolanza et al. found that asingle intravenous injection of 5×10¹² vg of rAAV6-sh1FRG1 rescuesmuscle histopathology and muscle function of FRG1 mice. In detail, 200μl containing 2×10¹² or 5×10¹² vg of vector in physiological solutionwere injected into the tail vein using a 25-gauge Terumo syringe. Themethod of Bortolanza et al. may be applied to an AAV expressing CRISPRCas and injected into humans at a dosage of about 2×10¹⁵ or 2×10¹⁶ vg ofvector.

Dumonceaux et al. (Molecular Therapy vol. 18 no. 5,881-887 May 2010)inhibit the myostatin pathway using the technique of RNA interferencedirected against the myostatin receptor AcvRIIb mRNA (sh-AcvRIIb). Therestoration of a quasi-dystrophin was mediated by the vectorized U7exon-skipping technique (U7-DYS). Adeno-associated vectors carryingeither the sh-AcvrIIb construct alone, the U7-DYS construct alone, or acombination of both constructs were injected in the tibialis anterior(TA) muscle of dystrophic mdx mice. The injections were performed with10¹¹ AAV viral genomes. The method of Dumonceaux et al. may be appliedto an AAV expressing CRISPR Cas and injected into humans, for example,at a dosage of about 10¹⁴ to about 10¹⁵ vg of vector.

Kinouchi et al. (Gene Therapy (2008) 15, 1126-1130) report theeffectiveness of in vivo siRNA delivery into skeletal muscles of normalor diseased mice through nanoparticle formation of chemically unmodifiedsiRNAs with atelocollagen (ATCOL). ATCOL-mediated local application ofsiRNA targeting myostatin, a negative regulator of skeletal musclegrowth, in mouse skeletal muscles or intravenously, caused a markedincrease in the muscle mass within a few weeks after application. Theseresults imply that ATCOL-mediated application of siRNAs is a powerfultool for future therapeutic use for diseases including muscular atrophy.MstsiRNAs (final concentration, 10 mM) were mixed with ATCOL (finalconcentration for local administration, 0.5%) (AteloGene, Kohken, Tokyo,Japan) according to the manufacturer's instructions. After anesthesia ofmice (20-week-old male C57BL/6) by Nembutal (25 mg/kg, i.p.), theMst-siRNA/ATCOL complex was injected into the masseter and bicepsfemoris muscles. The method of Kinouchi et al. may be applied to CRISPRCas and injected into a human, for example, at a dosage of about 500 to1000 ml of a 40 μM solution into the muscle. Hagstrom et al. (MolecularTherapy Vol. 10, No. 2, August 2004) describe an intravascular, nonviralmethodology that enables efficient and repeatable delivery of nucleicacids to muscle cells (myofibers) throughout the limb muscles ofmammals. The procedure involves the injection of naked plasmid DNA orsiRNA into a distal vein of a limb that is transiently isolated by atourniquet or blood pressure cuff. Nucleic acid delivery to myofibers isfacilitated by its rapid injection in sufficient volume to enableextravasation of the nucleic acid solution into muscle tissue. Highlevels of transgene expression in skeletal muscle were achieved in bothsmall and large animals with minimal toxicity. Evidence of siRNAdelivery to limb muscle was also obtained. For plasmid DNA intravenousinjection into a rhesus monkey, a threeway stopcock was connected to twosyringe pumps (Model PHD 2000; Harvard Instruments), each loaded with asingle syringe. Five minutes after a papaverine injection, pDNA (15.5 to25.7 mg in 40-100 ml saline) was injected at a rate of 1.7 or 2.0 ml/s.This could be scaled up for plasmid DNA expressing CRISPR Cas of thepresent invention with an injection of about 300 to 500 mg in 800 to2000 ml saline for a human. For adenoviral vector injections into a rat,2×10⁹ infectious particles were injected in 3 ml of normal salinesolution (NSS). This could be scaled up for an adenoviral vectorexpressing CRISPR Cas of the present invention with an injection ofabout 1×10¹³ infectious particles were injected in 10 liters of NSS fora human. For siRNA, a rat was injected into the great saphenous veinwith 12.5 μg of a siRNA and a primate was injected injected into thegreat saphenous vein with 750 μg of a siRNA. This could be scaled up fora CRISPR Cas of the present invention, for example, with an injection ofabout 15 to about 50 mg into the great saphenous vein of a human.

See also, for example, WO2013163628 A2, Genetic Correction of MutatedGenes, published application of Duke University describes efforts tocorrect, for example, a frameshift mutation which causes a prematurestop codon and a truncated gene product that can be corrected vianuclease mediated non-homologous end joining such as those responsiblefor Duchenne Muscular Dystrophy, (“DMD”) a recessive, fatal, X-linkeddisorder that results in muscle degeneration due to mutations in thedystrophin gene. The majority of dystrophin mutations that cause DMD aredeletions of exons that disrupt the reading frame and cause prematuretranslation termination in the dystrophin gene. Dystrophin is acytoplasmic protein that provides structural stability to thedystroglycan complex of the cell membrane that is responsible forregulating muscle cell integrity and function. The dystrophin gene or“DMD gene” as used interchangeably herein is 2.2 megabases at locusXp21. The primary transcription measures about 2,400 kb with the maturemRNA being about 14 kb. 79 exons code for the protein which is over 3500amino acids. Exon 51 is frequently adjacent to frame-disruptingdeletions in DMD patients and has been targeted in clinical trials foroligonucleotide-based exon skipping. A clinical trial for the exon 51skipping compound eteplirsen recently reported a significant functionalbenefit across 48 weeks, with an average of 47% dystrophin positivefibers compared to baseline. Mutations in exon 51 are ideally suited forpermanent correction by NHEJ-based genome editing.

The methods of US Patent Publication No. 20130145487 assigned toCellectis, which relates to meganuclease variants to cleave a targetsequence from the human dystrophin gene (DMD), may also be modified tofor the nucleic acid-targeting system of the present invention.

Treating Diseases of the Skin

The present invention also contemplates delivering the CRISPR-Cas systemdescribed herein, e.g. Cpf1 effector protein systems, to the skin.

Hickerson et al. (Molecular Therapy—Nucleic Acids (2013) 2, e129)relates to a motorized microneedle array skin delivery device fordelivering self-delivery (sd)-siRNA to human and murine skin. Theprimary challenge to translating siRNA-based skin therapeutics to theclinic is the development of effective delivery systems. Substantialeffort has been invested in a variety of skin delivery technologies withlimited success. In a clinical study in which skin was treated withsiRNA, the exquisite pain associated with the hypodermic needleinjection precluded enrollment of additional patients in the trial,highlighting the need for improved, more “patient-friendly” (i.e.,little or no pain) delivery approaches. Microneedles represent anefficient way to deliver large charged cargos including siRNAs acrossthe primary barrier, the stratum corneum, and are generally regarded asless painful than conventional hypodermic needles. Motorized “stamptype” microneedle devices, including the motorized microneedle array(MMNA) device used by Hickerson et al., have been shown to be safe inhairless mice studies and cause little or no pain as evidenced by (i)widespread use in the cosmetic industry and (ii) limited testing inwhich nearly all volunteers found use of the device to be much lesspainful than a flushot, suggesting siRNA delivery using this device willresult in much less pain than was experienced in the previous clinicaltrial using hypodermic needle injections. The MMNA device (marketed asTriple-M or Tri-M by Bomtech Electronic Co, Seoul, South Korea) wasadapted for delivery of siRNA to mouse and human skin. sd-siRNA solution(up to 300 μl of 0.1 mg/ml RNA) was introduced into the chamber of thedisposable Tri-M needle cartridge (Bomtech), which was set to a depth of0.1 mm. For treating human skin, deidentified skin (obtained immediatelyfollowing surgical procedures) was manually stretched and pinned to acork platform before treatment. All intradermal injections wereperformed using an insulin syringe with a 28-gauge 0.5-inch needle. TheMMNA device and method of Hickerson et al. could be used and/or adaptedto deliver the CRISPR Cas of the present invention, for example, at adosage of up to 300 μl of 0.1 mg/ml CRISPR Cas to the skin.

Leachman et al. (Molecular Therapy, vol. 18 no. 2, 442-446 February2010) relates to a phase Ib clinical trial for treatment of a rare skindisorder pachyonychia congenita (PC), an autosomal dominant syndromethat includes a disabling plantar keratoderma, utilizing the firstshort-interfering RNA (siRNA)-based therapeutic for skin. This siRNA,called TD101, specifically and potently targets the keratin 6a (K6a)N171K mutant mRNA without affecting wild-type K6a mRNA.

Zheng et al. (PNAS, Jul. 24, 2012, vol. 109, no. 30, 11975-11980) showthat spherical nucleic acid nanoparticle conjugates (SNA-NCs), goldcores surrounded by a dense shell of highly oriented, covalentlyimmobilized siRNA, freely penetrate almost 100% of keratinocytes invitro, mouse skin, and human epidermis within hours after application.Zheng et al. demonstrated that a single application of 25 nM epidermalgrowth factor receptor (EGFR) SNA-NCs for 60 h demonstrate effectivegene knockdown in human skin. A similar dosage may be contemplated forCRISPR Cas immobilized in SNA-NCs for administration to the skin.

Cancer

In some embodiments, the treatment, prophylaxis or diagnosis of canceris provided. The target is preferably one or more of the FAS, BID,CTLA4, PDCD1, CBLB, PTPN6, TRAC or TRBC genes. The cancer may be one ormore of lymphoma, chronic lymphocytic leukemia (CLL), B cell acutelymphocytic leukemia (B-ALL), acute lymphoblastic leukemia, acutemyeloid leukemia, non-Hodgkin's lymphoma (NHL), diffuse large celllymphoma (DLCL), multiple myeloma, renal cell carcinoma (RCC),neuroblastoma, colorectal cancer, breast cancer, ovarian cancer,melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer,hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma,head and neck cancer, and medulloblastoma. This may be implemented withengineered chimeric antigen receptor (CAR) T cell. This is described inWO2015161276, the disclosure of which is hereby incorporated byreference and described herein below.

Target genes suitable for the treatment or prophylaxis of cancer mayinclude, in some embodiments, those described in WO2015048577 thedisclosure of which is hereby incorporated by reference.

USHER Syndrome or Retinitis Pigmentosa-39

In some embodiments, the treatment, prophylaxis or diagnosis of UsherSyndrome or retinitis pigmentosa-39 is provided. The target ispreferably the USH2A gene. In some embodiments, correction of a Gdeletion at position 2299 (2299delG) is provided. This is described inWO2015134812A1, the disclosure of which is hereby incorporated byreference.

Cystic Fibrosis (CF)

In some embodiments, the treatment, prophylaxis or diagnosis of cysticfibrosis is provided. The target is preferably the SCNN1A or the CFTRgene. This is described in WO2015157070, the disclosure of which ishereby incorporated by reference.

Schwank et al. (Cell Stem Cell, 13:653-58, 2013) used CRISPR-Cas9 tocorrect a defect associated with cystic fibrosis in human stem cells.The team's target was the gene for an ion channel, cystic fibrosistransmembrane conductor receptor (CFTR). A deletion in CFTR causes theprotein to misfold in cystic fibrosis patients. Using culturedintestinal stem cells developed from cell samples from two children withcystic fibrosis, Schwank et al. were able to correct the defect usingCRISPR along with a donor plasmid containing the reparative sequence tobe inserted. The researchers then grew the cells into intestinal“organoids,” or miniature guts, and showed that they functionednormally. In this case, about half of clonal organoids underwent theproper genetic correction.

HIV and AIDS

In some embodiments, the treatment, prophylaxis or diagnosis of HIV andAIDS is provided. The target is preferably the CCR5 gene in HIV. This isdescribed in WO2015148670A1, the disclosure of which is herebyincorporated by reference.

Beta Thalassaemia

In some embodiments, the treatment, prophylaxis or diagnosis of BetaThalassaemia is provided. The target is preferably the BCL11A gene. Thisis described in WO2015148860, the disclosure of which is herebyincorporated by reference.

Sickle Cell Disease (SCD)

In some embodiments, the treatment, prophylaxis or diagnosis of SickleCell Disease (SCD) is provided. The target is preferably the HBB orBCL11A gene. This is described in WO2015148863, the disclosure of whichis hereby incorporated by reference.

Herpes Simplex Virus 1 and 2

In some embodiments, the treatment, prophylaxis or diagnosis of HSV-1(Herpes Simplex Virus 1) is provided. The target is preferably the UL19,UL30, UL48 or UL50 gene in HSV-1. This is described in WO2015153789, thedisclosure of which is hereby incorporated by reference.

In other embodiments, the treatment, prophylaxis or diagnosis of HSV-2(Herpes Simplex Virus 2) is provided. The target is preferably the UL19,UL30, UL48 or UL50 gene in HSV-2. This is described in WO2015153791, thedisclosure of which is hereby incorporated by reference.

In some embodiments, the treatment, prophylaxis or diagnosis of PrimaryOpen Angle Glaucoma (POAG) is provided. The target is preferably theMYOC gene. This is described in WO2015153780, the disclosure of which ishereby incorporated by reference.

Adoptive Cell Therapies

The present invention also contemplates use of the CRISPR-Cas systemdescribed herein, e.g. Cpf1 effector protein systems, to modify cellsfor adoptive therapies. Aspects of the invention accordingly involve theadoptive transfer of immune system cells, such as T cells, specific forselected antigens, such as tumor associated antigens (see Maus et al.,2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Review ofImmunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015, Adoptive celltransfer as personalized immunotherapy for human cancer, Science Vol.348 no. 6230 pp. 62-68; and, Restifo et al., 2015, Adoptiveimmunotherapy for cancer: harnessing the T cell response. Nat. Rev.Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Design andimplementation of adoptive therapy with chimeric antigenreceptor-modified T cells. Immunol Rev. 257(1): 127-144). Variousstrategies may for example be employed to genetically modify T cells byaltering the specificity of the T cell receptor (TCR) for example byintroducing new TCR α and β chains with selected peptide specificity(see U.S. Pat. No. 8,697,854; PCT Patent Publications: WO2003020763,WO2004033685, WO2004044004, WO2005114215, WO2006000830, WO2008038002,WO2008039818, WO2004074322, WO2005113595, WO2006125962, WO2013166321,WO2013039889, WO2014018863, WO2014083173; U.S. Pat. No. 8,088,379).

As an alternative to, or addition to, TCR modifications, chimericantigen receptors (CARs) may be used in order to generateimmunoresponsive cells, such as T cells, specific for selected targets,such as malignant cells, with a wide variety of receptor chimeraconstructs having been described (see U.S. Pat. Nos. 5,843,728;5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014;6,753,162; 8,211,422; and, PCT Publication WO9215322). Alternative CARconstructs may be characterized as belonging to successive generations.First-generation CARs typically consist of a single-chain variablefragment of an antibody specific for an antigen, for example comprisinga VL linked to a VH of a specific antibody, linked by a flexible linker,for example by a CD8α hinge domain and a CD8α transmembrane domain, tothe transmembrane and intracellular signaling domains of either CD3ζ orFcRγ (scFv-CD3ζ or scFv-FcRγ; see U.S. Pat. Nos. 7,741,465; 5,912,172;5,906,936). Second-generation CARs incorporate the intracellular domainsof one or more costimulatory molecules, such as CD28, OX40 (CD134), or4-1BB (CD137) within the endodomain (for examplescFv-CD28/OX40/4-1BB-CD3; see U.S. Pat. Nos. 8,911,993; 8,916,381;8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARsinclude a combination of costimulatory endodomains, such a CD3ζ-chain,CD97, GDI 1a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28signaling domains (for example scFv-CD28-4-1BB-CD3ζ orscFv-CD28-OX40-CD3ζ; see U.S. Pat. Nos. 8,906,682; 8,399,645; 5,686,281;PCT Publication No. WO2014134165; PCT Publication No. WO2012079000).Alternatively, costimulation may be orchestrated by expressing CARs inantigen-specific T cells, chosen so as to be activated and expandedfollowing engagement of their native αβTCR, for example by antigen onprofessional antigen-presenting cells, with attendant costimulation. Inaddition, additional engineered receptors may be provided on theimmunoresponsive cells, for example to improve targeting of a T-cellattack and/or minimize side effects.

Alternative techniques may be used to transform target immunoresponsivecells, such as protoplast fusion, lipofection, transfection orelectroporation. A wide variety of vectors may be used, such asretroviral vectors, lentiviral vectors, adenoviral vectors,adeno-associated viral vectors, plasmids or transposons, such as aSleeping Beauty transposon (see U.S. Pat. Nos. 6,489,458; 7,148,203;7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, forexample using 2nd generation antigen-specific CARs signaling throughCD3ζ and either CD28 or CD137. Viral vectors may for example includevectors based on HIV, SV40, EBV, HSV or BPV.

Cells that are targeted for transformation may for example include Tcells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL),regulatory T cells, human embryonic stem cells, tumor-infiltratinglymphocytes (TIL) or a pluripotent stem cell from which lymphoid cellsmay be differentiated. T cells expressing a desired CAR may for examplebe selected through co-culture with γ-irradiated activating andpropagating cells (AaPC), which co-express the cancer antigen andco-stimulatory molecules. The engineered CAR T-cells may be expanded,for example by co-culture on AaPC in presence of soluble factors, suchas IL-2 and IL-21. This expansion may for example be carried out so asto provide memory CAR+ T cells (which may for example be assayed bynon-enzymatic digital array and/or multi-panel flow cytometry). In thisway, CAR T cells may be provided that have specific cytotoxic activityagainst antigen-bearing tumors (optionally in conjunction withproduction of desired chemokines such as interferon-γ). CAR T cells ofthis kind may for example be used in animal models, for example tothreat tumor xenografts.

Approaches such as the foregoing may be adapted to provide methods oftreating and/or increasing survival of a subject having a disease, suchas a neoplasia, for example by administering an effective amount of animmunoresponsive cell comprising an antigen recognizing receptor thatbinds a selected antigen, wherein the binding activates theimmunoreponsive cell, thereby treating or preventing the disease (suchas a neoplasia, a pathogen infection, an autoimmune disorder, or anallogeneic transplant reaction). Dosing in CAR T cell therapies may forexample involve administration of from 106 to 109 cells/kg, with orwithout a course of lymphodepletion, for example with cyclophosphamide.

In one embodiment, the treatment can be administrated into patientsundergoing an immunosuppressive treatment. The cells or population ofcells, may be made resistant to at least one immunosuppressive agent dueto the inactivation of a gene encoding a receptor for suchimmunosuppressive agent. Not being bound by a theory, theimmunosuppressive treatment should help the selection and expansion ofthe immunoresponsive or T cells according to the invention within thepatient.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The cells or population of cells may be administeredto a patient subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. Dosing in CAR T cell therapies may for exampleinvolve administration of from 10⁶ to 10⁹ cells/kg, with or without acourse of lymphodepletion, for example with cyclophosphamide. The cellsor population of cells can be administrated in one or more doses. Inanother embodiment, the effective amount of cells are administrated as asingle dose. In another embodiment, the effective amount of cells areadministrated as more than one dose over a period time. Timing ofadministration is within the judgment of managing physician and dependson the clinical condition of the patient. The cells or population ofcells may be obtained from any source, such as a blood bank or a donor.While individual needs vary, determination of optimal ranges ofeffective amounts of a given cell type for a particular disease orconditions are within the skill of one in the art. An effective amountmeans an amount which provides a therapeutic or prophylactic benefit.The dosage administrated will be dependent upon the age, health andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired.

In another embodiment, the effective amount of cells or compositioncomprising those cells are administrated parenterally. Theadministration can be an intravenous administration. The administrationcan be directly done by injection within a tumor.

To guard against possible adverse reactions, engineered immunoresponsivecells may be equipped with a transgenic safety switch, in the form of atransgene that renders the cells vulnerable to exposure to a specificsignal. For example, the herpes simplex viral thymidine kinase (TK) genemay be used in this way, for example by introduction into allogeneic Tlymphocytes used as donor lymphocyte infusions following stem celltransplantation (Greco, et al., Improving the safety of cell therapywith the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells,administration of a nucleoside prodrug such as ganciclovir or acyclovircauses cell death. Alternative safety switch constructs includeinducible caspase 9, for example triggered by administration of asmall-molecule dimerizer that brings together two nonfunctional icasp9molecules to form the active enzyme. A wide variety of alternativeapproaches to implementing cellular proliferation controls have beendescribed (see U.S. Patent Publication No. 20130071414; PCT PatentPublication WO2011146862; PCT Patent Publication WO2014011987; PCTPatent Publication WO2013040371; Zhou et al. BLOOD, 2014,123/25:3895-3905; Di Stasi et al., The New England Journal of Medicine2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)).

In a further refinement of adoptive therapies, genome editing with aCRISPR-Cas system as described herein may be used to tailorimmunoresponsive cells to alternative implementations, for exampleproviding edited CAR T cells (see Poirot et al., 2015, Multiplex genomeedited T-cell manufacturing platform for “off-the-shelf” adoptive T-cellimmunotherapies, Cancer Res 75 (18): 3853). For example,immunoresponsive cells may be edited to delete expression of some or allof the class of HLA type II and/or type I molecules, or to knockoutselected genes that may inhibit the desired immune response, such as thePD1 gene.

Cells may be edited using any CRISPR system and method of use thereof asdescribed herein. CRISPR systems may be delivered to an immune cell byany method described herein. In preferred embodiments, cells are editedex vivo and transferred to a subject in need thereof. Immunoresponsivecells, CAR T cells or any cells used for adoptive cell transfer may beedited. Editing may be performed to eliminate potential alloreactiveT-cell receptors (TCR), disrupt the target of a chemotherapeutic agent,block an immune checkpoint, activate a T cell, and/or increase thedifferentiation and/or proliferation of functionally exhausted ordysfunctional CD8+ T-cells (see PCT Patent Publications: WO2013176915,WO2014059173, WO2014172606, WO2014184744, and WO2014191128). Editing mayresult in inactivation of a gene.

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In a particular embodiment, theCRISPR system specifically catalyzes cleavage in one targeted genethereby inactivating said targeted gene. The nucleic acid strand breakscaused are commonly repaired through the distinct mechanisms ofhomologous recombination or non-homologous end joining (NHEJ). However,NHEJ is an imperfect repair process that often results in changes to theDNA sequence at the site of the cleavage. Repair via non-homologous endjoining (NHEJ) often results in small insertions or deletions (Indel)and can be used for the creation of specific gene knockouts. Cells inwhich a cleavage induced mutagenesis event has occurred can beidentified and/or selected by well-known methods in the art.

T cell receptors (TCR) are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, α and β, which assemble toform a heterodimer and associates with the CD3-transducing subunits toform the T cell receptor complex present on the cell surface. Each α andβ chain of the TCR consists of an immunoglobulin-like N-terminalvariable (V) and constant (C) region, a hydrophobic transmembranedomain, and a short cytoplasmic region. As for immunoglobulin molecules,the variable region of the α and β chains are generated by V(D)Jrecombination, creating a large diversity of antigen specificitieswithin the population of T cells. However, in contrast toimmunoglobulins that recognize intact antigen, T cells are activated byprocessed peptide fragments in association with an WIC molecule,introducing an extra dimension to antigen recognition by T cells, knownas MHC restriction. Recognition of MHC disparities between the donor andrecipient through the T cell receptor leads to T cell proliferation andthe potential development of graft versus host disease (GVHD). Theinactivation of TCRα or TCRβ can result in the elimination of the TCRfrom the surface of T cells preventing recognition of alloantigen andthus GVHD. However, TCR disruption generally results in the eliminationof the CD3 signaling component and alters the means of further T cellexpansion.

Allogeneic cells are rapidly rejected by the host immune system. It hasbeen demonstrated that, allogeneic leukocytes present in non-irradiatedblood products will persist for no more than 5 to 6 days (Boni, Muranskiet al. 2008 Blood 1; 112(12):4746-54). Thus, to prevent rejection ofallogeneic cells, the host's immune system usually has to be suppressedto some extent. However, in the case of adoptive cell transfer the useof immunosuppressive drugs also have a detrimental effect on theintroduced therapeutic T cells. Therefore, to effectively use anadoptive immunotherapy approach in these conditions, the introducedcells would need to be resistant to the immunosuppressive treatment.Thus, in a particular embodiment, the present invention furthercomprises a step of modifying T cells to make them resistant to animmunosuppressive agent, preferably by inactivating at least one geneencoding a target for an immunosuppressive agent. An immunosuppressiveagent is an agent that suppresses immune function by one of severalmechanisms of action. An immunosuppressive agent can be, but is notlimited to a calcineurin inhibitor, a target of rapamycin, aninterleukin-2 receptor α-chain blocker, an inhibitor of inosinemonophosphate dehydrogenase, an inhibitor of dihydrofolic acidreductase, a corticosteroid or an immunosuppressive antimetabolite. Thepresent invention allows conferring immunosuppressive resistance to Tcells for immunotherapy by inactivating the target of theimmunosuppressive agent in T cells. As non-limiting examples, targetsfor an immunosuppressive agent can be a receptor for animmunosuppressive agent such as: CD52, glucocorticoid receptor (GR), aFKBP family gene member and a cyclophilin family gene member.

Immune checkpoints are inhibitory pathways that slow down or stop immunereactions and prevent excessive tissue damage from uncontrolled activityof immune cells. In certain embodiments, the immune checkpoint targetedis the programmed death-1 (PD-1 or CD279) gene (PDCD1). In otherembodiments, the immune checkpoint targeted is cytotoxicT-lymphocyte-associated antigen (CTLA-4). In additional embodiments, theimmune checkpoint targeted is another member of the CD28 and CTLA4 Igsuperfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additionalembodiments, the immune checkpoint targeted is a member of the TNFRsuperfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.

Additional immune checkpoints include Src homology 2 domain-containingprotein tyrosine phosphatase 1 (SHP-1) (Watson H A, et al., SHP-1: thenext checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016Apr. 15; 44(2):356-62). SHP-1 is a widely expressed inhibitory proteintyrosine phosphatase (PTP). In T-cells, it is a negative regulator ofantigen-dependent activation and proliferation. It is a cytosolicprotein, and therefore not amenable to antibody-mediated therapies, butits role in activation and proliferation makes it an attractive targetfor genetic manipulation in adoptive transfer strategies, such aschimeric antigen receptor (CAR) T cells. Immune checkpoints may alsoinclude T cell immunoreceptor with Ig and ITIM domains(TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al., (2015) BeyondCTLA-4 and PD-1, the generation Z of negative checkpoint regulators.Front. Immunol. 6:418).

WO2014172606 relates to the use of MT1 and/or MT1 inhibitors to increaseproliferation and/or activity of exhausted CD8+ T-cells and to decreaseCD8+ T-cell exhaustion (e.g., decrease functionally exhausted orunresponsive CD8+ immune cells). In certain embodiments,metallothioneins are targeted by gene editing in adoptively transferredT cells.

In certain embodiments, targets of gene editing may be at least onetargeted locus involved in the expression of an immune checkpointprotein. Such targets may include, but are not limited to CTLA4, PPP2CA,PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2,BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4),TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS,TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA,IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1,BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, OX40,CD137, GITR, CD27, SHP-1 or TIM-3. In preferred embodiments, the genelocus involved in the expression of PD-1 or CTLA-4 genes is targeted. Inother preferred embodiments, combinations of genes are targeted, such asbut not limited to PD-1 and TIGIT.

In other embodiments, at least two genes are edited. Pairs of genes mayinclude, but are not limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 andTCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ,TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 andTCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 andTCRα, 2B4 and TCRβ.

Whether prior to or after genetic modification of the T cells, the Tcells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. Tcells can be expanded in vitro or in vivo.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See MOLECULARCLONING: A LABORATORY MANUAL, 2nd edition (1989) (Sambrook, Fritsch andManiatis); MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012)(Green and Sambrook); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1987) (F.M. Ausubel, et al. eds.); the series METHODS IN ENZYMOLOGY (AcademicPress, Inc.); PCR 2: A PRACTICAL APPROACH (1995) (M. J. MacPherson, B.D. Hames and G. R. Taylor eds.); ANTIBODIES, A LABORATORY MANUAL (1988)(Harlow and Lane, eds.); ANTIBODIES A LABORATORY MANUAL, 2nd edition(2013) (E. A. Greenfield ed.); and ANIMAL CELL CULTURE (1987) (R. I.Freshney, ed.).

The practice of the present invention employs, unless otherwiseindicated, conventional techniques for generation of geneticallymodified mice. See Marten H. Hofker and Jan van Deursen, TRANSGENICMOUSE METHODS AND PROTOCOLS, 2nd edition (2011).

Gene Drives

The present invention also contemplates use of the CRISPR-Cas systemdescribed herein, e.g. Cpf1 effector protein systems, to provideRNA-guided gene drives, for example in systems analogous to gene drivesdescribed in PCT Patent Publication WO 2015/105928. Systems of this kindmay for example provide methods for altering eukaryotic germline cells,by introducing into the germline cell a nucleic acid sequence encodingan RNA-guided DNA nuclease and one or more guide RNAs. The guide RNAsmay be designed to be complementary to one or more target locations ongenomic DNA of the germline cell. The nucleic acid sequence encoding theRNA guided DNA nuclease and the nucleic acid sequence encoding the guideRNAs may be provided on constructs between flanking sequences, withpromoters arranged such that the germline cell may express the RNAguided DNA nuclease and the guide RNAs, together with any desiredcargo-encoding sequences that are also situated between the flankingsequences. The flanking sequences will typically include a sequencewhich is identical to a corresponding sequence on a selected targetchromosome, so that the flanking sequences work with the componentsencoded by the construct to facilitate insertion of the foreign nucleicacid construct sequences into genomic DNA at a target cut site bymechanisms such as homologous recombination, to render the germline cellhomozygous for the foreign nucleic acid sequence. In this way,gene-drive systems are capable of introgressing desired cargo genesthroughout a breeding population (Gantz et al., 2015, Highly efficientCas9-mediated gene drive for population modification of the malariavector mosquito Anopheles stephensi, PNAS 2015, published ahead of printNov. 23, 2015, doi:10.1073/pnas.1521077112; Esvelt et al., 2014,Concerning RNA-guided gene drives for the alteration of wild populationseLife 2014; 3:e03401). In select embodiments, target sequences may beselected which have few potential off-target sites in a genome.Targeting multiple sites within a target locus, using multiple guideRNAs, may increase the cutting frequency and hinder the evolution ofdrive resistant alleles. Truncated guide RNAs may reduce off-targetcutting. Paired nickases may be used instead of a single nuclease, tofurther increase specificity. Gene drive constructs may include cargosequences encoding transcriptional regulators, for example to activatehomologous recombination genes and/or repress non-homologousend-joining. Target sites may be chosen within an essential gene, sothat non-homologous end-joining events may cause lethality rather thancreating a drive-resistant allele. The gene drive constructs can beengineered to function in a range of hosts at a range of temperatures(Cho et al. 2013, Rapid and Tunable Control of Protein Stability inCaenorhabditis elegans Using a Small Molecule, PLoS ONE 8(8): e72393.doi:10.1371/journal.pone.0072393).

Xenotransplantation

The present invention also contemplates use of the CRISPR-Cas systemdescribed herein, e.g. Cpf1 effector protein systems, to provideRNA-guided DNA nucleases adapted to be used to provide modified tissuesfor transplantation. For example, RNA-guided DNA nucleases may be usedto knockout, knockdown or disrupt selected genes in an animal, such as atransgenic pig (such as the human heme oxygenase-1 transgenic pig line),for example by disrupting expression of genes that encode epitopesrecognized by the human immune system, i.e. xenoantigen genes. Candidateporcine genes for disruption may for example includea(1,3)-galactosyltransferase and cytidinemonophosphate-N-acetylneuraminic acid hydroxylase genes (see PCT PatentPublication WO 2014/066505). In addition, genes encoding endogenousretroviruses may be disrupted, for example the genes encoding allporcine endogenous retroviruses (see Yang et al., 2015, Genome-wideinactivation of porcine endogenous retroviruses (PERVs), Science 27 Nov.2015: Vol. 350 no. 6264 pp. 1101-1104). In addition, RNA-guided DNAnucleases may be used to target a site for integration of additionalgenes in xenotransplant donor animals, such as a human CD55 gene toimprove protection against hyperacute rejection.

General Gene Therapy Considerations

Examples of disease-associated genes and polynucleotides and diseasespecific information is available from McKusick-Nathans Institute ofGenetic Medicine, Johns Hopkins University (Baltimore, Md.) and NationalCenter for Biotechnology Information, National Library of Medicine(Bethesda, Md.), available on the World Wide Web.

Mutations in these genes and pathways can result in production ofimproper proteins or proteins in improper amounts which affect function.Further examples of genes, diseases and proteins are hereby incorporatedby reference from U.S. Provisional application 61/736,527 filed Dec. 12,2012. Such genes, proteins and pathways may be the target polynucleotideof a CRISPR complex of the present invention. Examples ofdisease-associated genes and polynucleotides are listed in Tables A andB. Examples of signaling biochemical pathway-associated genes andpolynucleotides are listed in Table C.

TABLE A DISEASE/DISORDERS GENE(S) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2;ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; ART; AKT2; AKT3; HIF;HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor);FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB(retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (Androgen Receptor);TSG101; IGF; IGF Receptor; Igf1 (4 variants); Igf2 (3 variants); Igf 1Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2,3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Macular Abcr; Ccl2; Cc2; cp(ceruloplasmin); Timp3; cathepsinD; Degeneration Vldlr; Ccr2Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin);Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophanhydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT (Slc6a4);COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1) Trinucleotide RepeatHTT (Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Disorders Dx); FXN/X25(Friedrich's Ataxia); ATX3 (Machado- Joseph's Dx); ATXN1 and ATXN2(spinocerebellar ataxias); DMPK (myotonic dystrophy); Atrophin-1 andAtn1 (DRPLA Dx); CBP (Creb-BP - global instability); VLDLR(Alzheimer's); Atxn7; Atxn10 Fragile X Syndrome FMR2; FXR1; FXR2; mGLUR5Secretase Related APH-1 (alpha and beta); Presenilin (Psen1); nicastrinDisorders (Ncstn); PEN-2 Others Nos1; Parp1; Nat1; Nat2 Prion - relateddisorders Prp ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b;VEGF-c) Drug addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol);GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) AutismMecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1;FXR2; Mglur5) Alzheimer's Disease E1; CHIP; UCH; UBB; Tau; LRP; PICALM;Clusterin; PS1; SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin1); Uchl1; Uchl3; APP Inflammation IL-10; IL-1 (IL-1a; IL-1b); IL-13;IL-17 (IL-17a (CTLA8); IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1;ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4;Cx3cl1 Parkinson's Disease x-Synuclein; DJ-1; LRRK2; Parkin; PINK1

TABLE B Blood and Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3,UMPH1, coagulation diseases PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2,ANH1, ASB, and disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome(TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP,RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H and factorH-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VIIdeficiency (F7); Factor X deficiency (F10); Factor XI deficiency (F11);Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A);Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA,FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1,FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocyticlymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3,HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB),Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies anddisorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3,EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia(HBA2, HBB, HBD, LCRB, HBA1). Cell dysregulation B-cell non-Hodgkinlymphoma (BCL7A, BCL7); Leukemia (TAL1, and oncology TCL5, SCL, TAL2,FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases and disorders HOXD4,HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12,LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT,LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3,FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM,CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF,WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA,GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN,CAIN). Inflammation and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1,IFNG, CXCL12, immune related SDF1); Autoimmune lymphoproliferativesyndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A);Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1 (CCL5,SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF,CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G,AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG,HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI);Inflammation (IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8),IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa,NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1);Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS,SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG,SCIDX1, SCIDX, IMD4). Metabolic, liver, Amyloid neuropathy (TTR, PALB);Amyloidosis (APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN, FGA,LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, diseases and disorders CIRH1A,NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7);Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2,LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1,HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder(SCOD1, SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancerand carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53,P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidneydisease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1,QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1,ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).Muscular/Skeletal Becker muscular dystrophy (DMD, BMD, MYF6), DuchenneMuscular diseases and disorders Dystrophy (DMD, BMD); Emery-Dreifussmuscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA,LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy(FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM,LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B,SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E,SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H,FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C,SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1,LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7,OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2,SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2,CATF1, SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP,VEGF (VEGF-a, VEGF-b, neuronal diseases and VEGF-c); Alzheimer disease(APP, AAA, CVAP, AD1, APOE, AD2, disorders PSEN2, AD4, STM2, APBB2,FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP,A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A,Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4,KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5);Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP,JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT,TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2,PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN,PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79,CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1);Schizophrenia (Neuregulin1 (Nrg1), Erb4 (receptor for Neuregulin),Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophanhydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD(Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders(APH-1 (alpha and beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2,Nos1, Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT(Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich'sAtaxia), ATX3 (Machado- Joseph's Dx), ATXN1 and ATXN2 (spinocerebellarataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP(Creb-BP - global instability), VLDLR (Alzheimer's), Atxn7, Atxn10).Occular diseases and Age-related macular degeneration (Aber, Ccl2, Cc2,cp (ceruloplasmin), disorders Timp3, cathepsinD, Vldlr, Ccr2); Cataract(CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1,PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD,CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2,CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA,CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1);Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3,CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD,PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma(MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1,GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1,RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4,GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4,ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

TABLE C CELLULAR FUNCTION GENES PI3K/AKT Signaling PRKCE; ITGAM; ITGA5;IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1;AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8;BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1;MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB;DYRK1A; CDKN1A; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1;PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN;ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2;RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA;CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8;MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9;SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1;FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3;ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF;STAT1; SGK Glucocorticoid Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6;PCAF; ELK1; Signaling MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA;CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8;BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A;MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3;MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8;NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1;SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 AxonalGuidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; Signaling IGF1;RAC1; RAP1A; EIF4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF;RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ;PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS;RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2;PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3;CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA EphrinReceptor PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2;EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1;AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8;GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2;PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; AKT1; JAK2;STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK;CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK Actin Cytoskeleton ACTN4;PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; INS;ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1;PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS;RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN;VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1;PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGKHuntington's Disease PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2;Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5;CREB1; PRKCI; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1;GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11;MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1;CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK;HDAC6; CASP3 Apoptosis Signaling PRKCE; ROCK1; BID; IRAK1; PRKAA2;EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2;CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8;KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG;RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA;CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 BCell Receptor RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; SignalingAKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3;MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9;EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1;PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN;GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte Extravasation ACTN4; CD44;PRKCE; ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A; PRKCZ; ROCK2;RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8;PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A;BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1;CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9Integrin Signaling ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1;ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3;MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7;PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1;TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11;Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8;RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1;TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2;AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3;IL1R1; IL6 PTEN Signaling ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11;MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2;PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1;IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1;MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1;CASP3; RPS6KB1 p53 Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1;GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3;MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1;HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1;RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2;GSK3B; BAχ; AKT3 Aryl Hydrocarbon HSPB1; EP300; FASN; TGM2; RXRA; MAPK1;NQO1; Receptor Signaling NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; SMARCA4;NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73;GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2;APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6;CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE; EP300; PRKCZ; RXRA; MAPK1;NQO1; Signaling NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB;PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13;PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A;PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1;NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK SignalingPRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2;PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1;IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1;PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3;CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling PRKAA2;EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB;NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS;RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7;CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1;PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB Signaling IRAK1; EIF2AK2; EP300; INS;MYD88; PRKCZ; TRAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2;MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A;TRAF2; TLR4; PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1;PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1Neuregulin Signaling ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1;MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3;ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17;AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC;NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44;EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC;GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2; ILK; LEF1;SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1;TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2Insulin Receptor PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; SignalingPTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3;TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2;JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B;AKT3; FOXO1; SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1;MAPK1; PTPN11; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST;KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1;IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1;CEBPB; JUN; IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS;MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8;PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG;RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN;IL1R1; PRKCA; IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11;NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R;IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2;AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF;CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1;Oxidative Stress Response NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A;MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN;KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic Fibrosis/HepaticEDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; Stellate Cell ActivationSMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4;PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1;CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS;TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B;MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF;INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1;NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon RI Signaling PRKCE; RAC1; PRKCZ;LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK;MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3;PRKCA G-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB;Receptor Signaling PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3;MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1;PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCAInositol Phosphate PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MetabolismMAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD;PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1;MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1;ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3;KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA;STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGFSignaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA;ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3;PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA;AKT3; FOXO1; PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2;PTPN11; Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4;AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4;SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation ATR; ABL1;E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53;CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1;HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS;Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA; PIK3C2A; BTK;LCK; RAF1; IKBKG; RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK;BCL10; JUN; VAV3 Death Receptor Signaling CRADD; HSPB1; BID; BIRC4;TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX;TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1;CASP2; BIRC2; CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2;MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3;MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1;FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GM-CSF Signaling LYN; ELK1; MAPK1;PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1;MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID; IGF1;RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA; BCL2;PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A;CASP1; APAF1; VEGFA; BIRC2; BAχ; AKT3; CASP3; BIRC3 JAK/Stat SignalingPTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS;SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1;PRKAA2; EIF2AK2; GRK6; MAPK1; Nicotinamide Metabolism PLK1; AKT2; CDK8;MAPK8; MAPK3; PRKCD; PRKAA1; PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2;MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine Signaling CXCR4;ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS;MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1;JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK;FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A;LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 Synaptic LongTerm PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1; GNAS; Depression PRKCI;GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A;PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA Estrogen ReceptorTAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; Signaling SMARCA4; MAPK3;NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP;MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 Protein Ubiquitination TRAF6;SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; Pathway CBL; UBE2I; BTRC; HSPA5;USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1;VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1; ELK1; IKBKB; SP1; FOS;NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7;JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXR Activation PRKCE;EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1;PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1;PRKCA TGF-beta Signaling EP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; FOS;MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1; RAF1; MAP3K7; CREBBP;MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Toll-like Receptor IRAK1;EIF2AK2; MYD88; TRAF6; PPARA; ELK1; Signaling IKBKB; FOS; NFKB2;MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK;NFKB1; TLR2; JUN p38 MAPK Signaling HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1;FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF;MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1 Neurotrophin/TRK NTRK2;MAPK1; PTPN11; PIK3CA; CREB1; FOS; Signaling PIK3CB; PIK3C3; MAPK8;MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42;JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8;APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1;FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE; RAP1A; EP300; PRKCZ; MAPK1;CREB1; Potentiation PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; PRKCD;PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4; PRKCA Calcium SignalingRAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2;HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; HDAC6 EGFSignaling ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3;PIK3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1Hypoxia Signaling in the EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT;Cardiovascular System HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; VEGFA;JUN; ATF4; VHL; HSP90AA1 LPS/IL-1 Mediated IRAK1; MYD88; TRAF6; PPARA;RXRA; ABCA1; Inhibition of RXR Function MAPK8; ALDH1A1; GSTP1; MAPK9;ABCB1; TRAF2; TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; IL1R1 LXR/RXRActivation FASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA; NOS2A; TLR4;TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 AmyloidProcessing PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3;MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP IL-4Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1; KRAS; SOCS1; PTPN6; NR3C1;PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KB1 Cell Cycle: G2/MDNA EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; Damage Checkpoint CHEK1;ATR; CHEK2; YWHAZ; TP53; CDKN1A; Regulation PRKDC; ATM; SFN; CDKN2ANitric Oxide Signaling in KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3;the Cardiovascular System CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1;VEGFA; AKT3; HSP90AA1 Purine Metabolism NME2; SMARCA4; MYH9; RRM2; ADAR;EIF2AK4; PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1cAMP-mediated RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3; Signaling SRC;RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8;CASP8; MAPK10; MAPK9; CASP9; Dysfunction PARK7; PSEN1; PARK2; APP; CASP3Notch Signaling HES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3;NOTCH1; DLL4 Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6;CASP9; ATF4; Stress Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2;AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson'sSignaling UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3Cardiac & Beta GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; AdrenergicSignaling PPP2R5C Glycolysis/Gluconeogenesis HK2; GCK; GPI; ALDH1A1;PKM2; LDHA; HK1 Interferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1;STAT1; IFIT3 Sonic Hedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B;DYRK1B Signaling Glycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1;SPHK2 Metabolism Phospholipid PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2Degradation Tryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1;SIAH1 Lysine Degradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C NucleotideExcision ERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and SucroseUCHL1; HK2; GCK; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2;GCK; HK1 Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism CircadianRhythm CSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1;F2R; SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5CSignaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1 GlycerolipidMetabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid PRDX6; GRN; YWHAZ;CYP1B1 Metabolism Methionine Metabolism DNMT1; DNMT3B; AHCY; DNMT3APyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and ProlineALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN; YWHAZFructose and Mannose HK2; GCK; HK1 Metabolism Galactose Metabolism HK2;GCK; HK1 Stilbene, Coumarine and PRDX6; PRDX1; TYR Lignin BiosynthesisAntigen Presentation CALR; B2M Pathway Biosynthesis of Steroids NQO1;DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 FattyAcid Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKAMetabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol MetabolismERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome p450Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1 MetabolismPropanoate Metabolism ALDH1A1; LDHA Selenoamino Acid PRMT5; AHCYMetabolism Sphingolipid Metabolism SPHK1; SPHK2 Aminophosphonate PRMT5Metabolism Androgen and Estrogen PRMT5 Metabolism Ascorbate and AldarateALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1 Cysteine MetabolismLDHA Fatty Acid Biosynthesis FASN Glutamate Receptor GNB2L1 SignalingNRF2-mediated PRDX1 Oxidative Stress Response Pentose Phosphate GPIPathway Pentose and Glucuronate UCHL1 Interconversions RetinolMetabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5,TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1 IsoleucineDegradation Glycine, Serine and CHKA Threonine Metabolism LysineDegradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6;TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5;Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF; CytC; SMAC(Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Noggin(Nog); WNT (Wnt2; Neurology Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b;Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1;Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86(Pou4f1 or Brn3a); Numb; Reln

Embodiments of the invention also relate to methods and compositionsrelated to knocking out genes, amplifying genes and repairing particularmutations associated with DNA repeat instability and neurologicaldisorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities andNeurological Diseases, Second Edition, Academic Press, Oct. 13,2011—Medical). Specific aspects of tandem repeat sequences have beenfound to be responsible for more than twenty human diseases (Newinsights into repeat instability: role of RNA.DNA hybrids. McIvor E I,Polak U, Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). Thepresent effector protein systems may be harnessed to correct thesedefects of genomic instability.

Several further aspects of the invention relate to correcting defectsassociated with a wide range of genetic diseases which are furtherdescribed on the website of the National Institutes of Health under thetopic subsection Genetic Disorders (website athealth.nih.gov/topic/GeneticDisorders). The genetic brain diseases mayinclude but are not limited to Adrenoleukodystrophy, Agenesis of theCorpus Callosum, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease,Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration,Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington'sDisease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-NyhanSyndrome, Menkes Disease, Mitochondrial Myopathies and NINDSColpocephaly. These diseases are further described on the website of theNational Institutes of Health under the subsection Genetic BrainDisorders.

Cas9 Development and Use

The present invention may be further illustrated and extended based onaspects of CRISPR-Cas9 development and use as set forth in the followingarticles and particularly as relates to delivery of a CRISPR proteincomplex and uses of an RNA guided endonuclease in cells and organisms:

-   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,    Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D.,    Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science February    15; 339(6121):819-23 (2013);-   RNA-guided editing of bacterial genomes using CRISPR-Cas systems.    Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol    March; 31(3):233-9 (2013);-   One-Step Generation of Mice Carrying Mutations in Multiple Genes by    CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila    C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9;    153(4):910-8 (2013);-   Optical control of mammalian endogenous transcription and epigenetic    states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich    M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. August    22; 500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug. 23    (2013);-   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing    Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S.,    Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S.,    Zhang, Y., & Zhang, F. Cell August 28. pii: S0092-8674(13)01015-5    (2013-A);-   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,    Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V.,    Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L    A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);-   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P    D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature    Protocols November; 8(11):2281-308 (2013-B);-   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem,    O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson,    T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F.    Science December 12. (2013). [Epub ahead of print];-   Crystal structure of cas9 in complex with guide RNA and target DNA.    Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I.,    Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27,    156(5):935-49 (2014);-   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian    cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D    B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R.,    Zhang F., Sharp P A. Nat Biotechnol. April 20. doi: 10.1038/nbt.2889    (2014);-   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.    Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R, Dahlman J    E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B, Jhunjhunwala    S, Heidenreich M, Xavier R J, Langer R, Anderson D G, Hacohen N,    Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2): 440-455 DOI:    10.1016/j.cell.2014.09.014 (2014);-   Development and Applications of CRISPR-Cas9 for Genome Engineering,    Hsu P D, Lander E S, Zhang F., Cell. June 5; 157(6):1262-78 (2014).-   Genetic screens in human cells using the CRISPR/Cas9 system, Wang T,    Wei J J, Sabatini D M, Lander E S., Science. January 3; 343(6166):    80-84. doi:10.1126/science.1246981 (2014);-   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated    gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova Z,    Hegde M, Smith I, Sullender M, Ebert B L,-   Xavier R J, Root D E., (published online 3 Sep. 2014) Nat    Biotechnol. December; 32(12):1262-7 (2014);-   In vivo interrogation of gene function in the mammalian brain using    CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y,    Trombetta J, Sur M, Zhang F., (published online 19 Oct. 2014) Nat    Biotechnol. January; 33(1):102-6 (2015);-   Genome-scale transcriptional activation by an engineered CRISPR-Cas9    complex, Konermann S, Brigham M D, Trevino A E, Joung J, Abudayyeh O    O, Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki    O, Zhang F., Nature. January 29; 517(7536):583-8 (2015).-   A split-Cas9 architecture for inducible genome editing and    transcription modulation, Zetsche B, Volz S E, Zhang F., (published    online 2 Feb. 2015) Nat Biotechnol. February; 33(2):139-42 (2015);-   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and    Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi X,    Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp P A.    Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen in mouse), and-   In vivo genome editing using Staphylococcus aureus Cas9, Ran F A,    Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche B,    Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang F.,    (published online 1 Apr. 2015), Nature. April 9; 520(7546):186-91    (2015).    -   Shalem et al., “High-throughput functional genomics using        CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015).    -   Xu et al., “Sequence determinants of improved CRISPR sgRNA        design,” Genome Research 25, 1147-1157 (August 2015).    -   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune        Cells to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul.        30, 2015).    -   Ramanan et al., CRISPR/Cas9 cleavage of viral DNA efficiently        suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:        10.1038/srep10833 (Jun. 2, 2015)    -   Nishimasu et al., Crystal Structure of Staphylococcus aureus        Cas9,” Cell 162, 1113-1126 (Aug. 27, 2015)    -   BCL11A enhancer dissection by Cas9-mediated in situ saturating        mutagenesis, Canver et al., Nature 527(7577):192-7 (Nov.        12, 2015) doi: 10.1038/nature15521. Epub 2015 Sep. 16.    -   Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas        System, Zetsche et al., Cell 163, 759-71 (Sep. 25, 2015).    -   Discovery and Functional Characterization of Diverse Class 2        CRISPR-Cas Systems, Shmakov et al., Molecular Cell, 60(3),        385-397 doi: 10.1016/j.molcel.2015.10.008 Epub Oct. 22, 2015.    -   Rationally engineered Cas9 nucleases with improved specificity,        Slaymaker et al., Science 2016 Jan. 1 351(6268): 84-88 doi:        10.1126/science.aad5227. Epub 2015 Dec. 1. [Epub ahead of        print].        each of which is incorporated herein by reference, may be        considered in the practice of the instant invention, and        discussed briefly below:-   Cong et al. engineered type II CRISPR-Cas systems for use in    eukaryotic cells based on both Streptococcus thermophilus Cas9 and    also Streptococcus pyogenes Cas9 and demonstrated that Cas9    nucleases can be directed by short RNAs to induce precise cleavage    of DNA in human and mouse cells. Their study further showed that    Cas9 as converted into a nicking enzyme can be used to facilitate    homology-directed repair in eukaryotic cells with minimal mutagenic    activity. Additionally, their study demonstrated that multiple guide    sequences can be encoded into a single CRISPR array to enable    simultaneous editing of several at endogenous genomic loci sites    within the mammalian genome, demonstrating easy programmability and    wide applicability of the RNA-guided nuclease technology. This    ability to use RNA to program sequence specific DNA cleavage in    cells defined a new class of genome engineering tools. These studies    further showed that other CRISPR loci are likely to be    transplantable into mammalian cells and can also mediate mammalian    genome cleavage. Importantly, it can be envisaged that several    aspects of the CRISPR-Cas system can be further improved to increase    its efficiency and versatility.-   Jiang et al. used the clustered, regularly interspaced, short    palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed    with dual-RNAs to introduce precise mutations in the genomes of    Streptococcus pneumoniae and Escherichia coli. The approach relied    on dual-RNA:Cas9-directed cleavage at the targeted genomic site to    kill unmutated cells and circumvents the need for selectable markers    or counter-selection systems. The study reported reprogramming    dual-RNA:Cas9 specificity by changing the sequence of short CRISPR    RNA (crRNA) to make single- and multinucleotide changes carried on    editing templates. The study showed that simultaneous use of two    crRNAs enabled multiplex mutagenesis. Furthermore, when the approach    was used in combination with recombineering, in S. pneumoniae,    nearly 100% of cells that were recovered using the described    approach contained the desired mutation, and in E. coli, 65% that    were recovered contained the mutation.-   Wang et al. (2013) used the CRISPR-Cas system for the one-step    generation of mice carrying mutations in multiple genes which were    traditionally generated in multiple steps by sequential    recombination in embryonic stem cells and/or time-consuming    intercrossing of mice with a single mutation. The CRISPR-Cas system    will greatly accelerate the in vivo study of functionally redundant    genes and of epistatic gene interactions.-   Konermann et al. (2013) addressed the need in the art for versatile    and robust technologies that enable optical and chemical modulation    of DNA-binding domains based CRISPR Cas9 enzyme and also    Transcriptional Activator Like Effectors-   Ran et al. (2013-A) described an approach that combined a Cas9    nickase mutant with paired guide RNAs to introduce targeted    double-strand breaks. This addresses the issue of the Cas9 nuclease    from the microbial CRISPR-Cas system being targeted to specific    genomic loci by a guide sequence, which can tolerate certain    mismatches to the DNA target and thereby promote undesired    off-target mutagenesis. Because individual nicks in the genome are    repaired with high fidelity, simultaneous nicking via appropriately    offset guide RNAs is required for double-stranded breaks and extends    the number of specifically recognized bases for target cleavage. The    authors demonstrated that using paired nicking can reduce off-target    activity by 50- to 1,500-fold in cell lines and to facilitate gene    knockout in mouse zygotes without sacrificing on-target cleavage    efficiency. This versatile strategy enables a wide variety of genome    editing applications that require high specificity.-   Hsu et al. (2013) characterized SpCas9 targeting specificity in    human cells to inform the selection of target sites and avoid    off-target effects. The study evaluated >700 guide RNA variants and    SpCas9-induced indel mutation levels at >100 predicted genomic    off-target loci in 293T and 293FT cells. The authors that SpCas9    tolerates mismatches between guide RNA and target DNA at different    positions in a sequence-dependent manner, sensitive to the number,    position and distribution of mismatches. The authors further showed    that SpCas9-mediated cleavage is unaffected by DNA methylation and    that the dosage of SpCas9 and gRNA can be titrated to minimize    off-target modification. Additionally, to facilitate mammalian    genome engineering applications, the authors reported providing a    web-based software tool to guide the selection and validation of    target sequences as well as off-target analyses.-   Ran et al. (2013-B) described a set of tools for Cas9-mediated    genome editing via non-homologous end joining (NHEJ) or    homology-directed repair (HDR) in mammalian cells, as well as    generation of modified cell lines for downstream functional studies.    To minimize off-target cleavage, the authors further described a    double-nicking strategy using the Cas9 nickase mutant with paired    guide RNAs. The protocol provided by the authors experimentally    derived guidelines for the selection of target sites, evaluation of    cleavage efficiency and analysis of off-target activity. The studies    showed that beginning with target design, gene modifications can be    achieved within as little as 1-2 weeks, and modified clonal cell    lines can be derived within 2-3 weeks.-   Shalem et al. described a new way to interrogate gene function on a    genome-wide scale. Their studies showed that delivery of a    genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted 18,080    genes with 64,751 unique guide sequences enabled both negative and    positive selection screening in human cells. First, the authors    showed use of the GeCKO library to identify genes essential for cell    viability in cancer and pluripotent stem cells. Next, in a melanoma    model, the authors screened for genes whose loss is involved in    resistance to vemurafenib, a therapeutic that inhibits mutant    protein kinase BRAF. Their studies showed that the highest-ranking    candidates included previously validated genes NF1 and MED12 as well    as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a    high level of consistency between independent guide RNAs targeting    the same gene and a high rate of hit confirmation, and thus    demonstrated the promise of genome-scale screening with Cas9.-   Nishimasu et al. reported the crystal structure of Streptococcus    pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°    resolution. The structure revealed a bilobed architecture composed    of target recognition and nuclease lobes, accommodating the    sgRNA:DNA heteroduplex in a positively charged groove at their    interface. Whereas the recognition lobe is essential for binding    sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease    domains, which are properly positioned for cleavage of the    complementary and non-complementary strands of the target DNA,    respectively. The nuclease lobe also contains a carboxyl-terminal    domain responsible for the interaction with the protospacer adjacent    motif (PAM). This high-resolution structure and accompanying    functional analyses have revealed the molecular mechanism of    RNA-guided DNA targeting by Cas9, thus paving the way for the    rational design of new, versatile genome-editing technologies.-   Wu et al. mapped genome-wide binding sites of a catalytically    inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single    guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). The    authors showed that each of the four sgRNAs tested targets dCas9 to    between tens and thousands of genomic sites, frequently    characterized by a 5-nucleotide seed region in the sgRNA and an NGG    protospacer adjacent motif (PAM). Chromatin inaccessibility    decreases dCas9 binding to other sites with matching seed sequences;    thus 70% of off-target sites are associated with genes. The authors    showed that targeted sequencing of 295 dCas9 binding sites in mESCs    transfected with catalytically active Cas9 identified only one site    mutated above background levels. The authors proposed a two-state    model for Cas9 binding and cleavage, in which a seed match triggers    binding but extensive pairing with target DNA is required for    cleavage.-   Platt et al. established a Cre-dependent Cas9 knockin mouse. The    authors demonstrated in vivo as well as ex vivo genome editing using    adeno-associated virus (AAV)-, lentivirus-, or particle-mediated    delivery of guide RNA in neurons, immune cells, and endothelial    cells.-   Hsu et al. (2014) is a review article that discusses generally    CRISPR-Cas9 history from yogurt to genome editing, including genetic    screening of cells.-   Wang et al. (2014) relates to a pooled, loss-of-function genetic    screening approach suitable for both positive and negative selection    that uses a genome-scale lentiviral single guide RNA (sgRNA)    library.-   Doench et al. created a pool of sgRNAs, tiling across all possible    target sites of a panel of six endogenous mouse and three endogenous    human genes and quantitatively assessed their ability to produce    null alleles of their target gene by antibody staining and flow    cytometry. The authors showed that optimization of the PAM improved    activity and also provided an on-line tool for designing sgRNAs.-   Swiech et al. demonstrate that AAV-mediated SpCas9 genome editing    can enable reverse genetic studies of gene function in the brain.-   Konermann et al. (2015) discusses the ability to attach multiple    effector domains, e.g., transcriptional activator, functional and    epigenomic regulators at appropriate positions on the guide such as    stem or tetraloop with and without linkers.-   Zetsche et al. demonstrates that the Cas9 enzyme can be split into    two and hence the assembly of Cas9 for activation can be controlled.-   Chen et al. relates to multiplex screening by demonstrating that a    genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes    regulating lung metastasis.-   Ran et al. (2015) relates to SaCas9 and its ability to edit genomes    and demonstrates that one cannot extrapolate from biochemical    assays.-   Shalem et al. (2015) described ways in which catalytically inactive    Cas9 (dCas9) fusions are used to synthetically repress (CRISPRi) or    activate (CRISPRa) expression, showing. advances using Cas9 for    genome-scale screens, including arrayed and pooled screens, knockout    approaches that inactivate genomic loci and strategies that modulate    transcriptional activity.-   Xu et al. (2015) assessed the DNA sequence features that contribute    to single guide RNA (sgRNA) efficiency in CRISPR-based screens. The    authors explored efficiency of CRISPR/Cas9 knockout and nucleotide    preference at the cleavage site. The authors also found that the    sequence preference for CRISPRi/a is substantially different from    that for CRISPR/Cas9 knockout.-   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9    libraries into dendritic cells (DCs) to identify genes that control    the induction of tumor necrosis factor (Tnf) by bacterial    lipopolysaccharide (LPS). Known regulators of Tlr4 signaling and    previously unknown candidates were identified and classified into    three functional modules with distinct effects on the canonical    responses to LPS.-   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA    (cccDNA) in infected cells. The HBV genome exists in the nuclei of    infected hepatocytes as a 3.2 kb double-stranded episomal DNA    species called covalently closed circular DNA (cccDNA), which is a    key component in the HBV life cycle whose replication is not    inhibited by current therapies. The authors showed that sgRNAs    specifically targeting highly conserved regions of HBV robustly    suppresses viral replication and depleted cccDNA.-   Nishimasu et al. (2015) reported the crystal structures of SaCas9 in    complex with a single guide RNA (sgRNA) and its double-stranded DNA    targets, containing the 5′-TTGAAT-3′ PAM and the 5′-TTGGGT-3′ PAM. A    structural comparison of SaCas9 with SpCas9 highlighted both    structural conservation and divergence, explaining their distinct    PAM specificities and orthologous sgRNA recognition.-   Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional    investigation of non-coding genomic elements. The authors we    developed pooled CRISPR-Cas9 guide RNA libraries to perform in situ    saturating mutagenesis of the human and mouse BCL11A enhancers which    revealed critical features of the enhancers.-   Zetsche et al. (2015) reported characterization of Cpf1, a class 2    CRISPR nuclease from Francisella novicida U112 having features    distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking    tracrRNA, utilizes a T-rich protospacer-adjacent motif, and cleaves    DNA via a staggered DNA double-stranded break.-   Shmakov et al. (2015) reported three distinct Class 2 CRISPR-Cas    systems. Two system CRISPR enzymes (C2c1 and C2c3) contain RuvC-like    endonuclease domains distantly related to Cpf1. Unlike Cpf1, C2c1    depends on both crRNA and tracrRNA for DNA cleavage. The third    enzyme (C2c2) contains two predicted HEPN RNase domains and is    tracrRNA independent.-   Slaymaker et al (2016) reported the use of structure-guided protein    engineering to improve the specificity of Streptococcus pyogenes    Cas9 (SpCas9). The authors developed “enhanced specificity” SpCas9    (eSpCas9) variants which maintained robust on-target cleavage with    reduced off-target effects.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

U.S. Pat. Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445,8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, 8,945,839,8,993,233 and 8,999,641; US Patent Publications US 2014-0310830 (U.S.application Ser. No. 14/105,031), US 2014-0287938 A1 (U.S. applicationSer. No. 14/213,991), US 2014-0273234 A1 (U.S. application Ser. No.14/293,674), US2014-0273232 A1 (U.S. application Ser. No. 14/290,575),US 2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046A1 (U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429); US 2015-0184139(U.S. application Ser. No. 14/324,960); Ser. No. 14/054,414 EuropeanPatent Applications EP 2 771 468 (EP13818570.7), EP 2 764 103(EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT PatentPublications WO 2014/093661 (PCT/US2013/074743), WO 2014/093694(PCT/US2013/074790), WO 2014/093595 (PCT/US2013/074611), WO 2014/093718(PCT/US2013/074825), WO 2014/093709 (PCT/US2013/074812), WO 2014/093622(PCT/US2013/074667), WO 2014/093635 (PCT/US2013/074691), WO 2014/093655(PCT/US2013/074736), WO 2014/093712 (PCT/US2013/074819), WO 2014/093701(PCT/US2013/074800), WO 2014/018423 (PCT/US2013/051418), WO 2014/204723(PCT/US2014/041790), WO 2014/204724 (PCT/US2014/041800), WO 2014/204725(PCT/US2014/041803), WO 2014/204726 (PCT/US2014/041804), WO 2014/204727(PCT/US2014/041806), WO 2014/204728 (PCT/US2014/041808), WO 2014/204729(PCT/US2014/041809), WO 2015/089351 (PCT/US2014/069897), WO 2015/089354(PCT/US2014/069902), WO 2015/089364 (PCT/US2014/069925), WO 2015/089427(PCT/US2014/070068), WO 2015/089462 (PCT/US2014/070127), WO 2015/089419(PCT/US2014/070057), WO 2015/089465 (PCT/US2014/070135), WO 2015/089486(PCT/US2014/070175), PCT/US2015/051691, PCT/US2015/051830. Reference isalso made to U.S. provisional patent applications 61/758,468;61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130, filed onJan. 30, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013and May 28, 2013 respectively. Reference is also made to U.S.provisional patent application 61/836,123, filed on Jun. 17, 2013.Reference is additionally made to U.S. provisional patent applications61/835,931, 61/835,936, 61/835,973, 61/836,080, 61/836,101, and61/836,127, each filed Jun. 17, 2013. Further reference is made to U.S.provisional patent applications 61/862,468 and 61/862,355 filed on Aug.5, 2013; 61/871,301 filed on Aug. 28, 2013; 61/960,777 filed on Sep. 25,2013 and 61/961,980 filed on Oct. 28, 2013. Reference is yet furthermade to: PCT/US2014/62558 filed Oct. 28, 2014, and U.S. ProvisionalPatent Applications Ser. Nos. 61/915,148, 61/915,150, 61/915,153,61/915,203, 61/915,251, 61/915,301, 61/915,267, 61/915,260, and61/915,397, each filed Dec. 12, 2013; 61/757,972 and 61/768,959, filedon Jan. 29, 2013 and Feb. 25, 2013; 62/010,888 and 62/010,879, bothfiled Jun. 11, 2014; 62/010,329, 62/010,439 and 62/010,441, each filedJun. 10, 2014; 61/939,228 and 61/939,242, each filed Feb. 12, 2014;61/980,012, filed Apr. 15, 2014; 62/038,358, filed Aug. 17, 2014;62/055,484, 62/055,460 and 62/055,487, each filed Sep. 25, 2014; and62/069,243, filed Oct. 27, 2014. Reference is made to PCT applicationdesignating, inter alia, the United States, application No.PCT/US14/41806, filed Jun. 10, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.Reference is made to PCT application designating, inter alia, the UnitedStates, application No. PCT/US14/41806, filed Jun. 10, 2014.

Mention is also made of U.S. application 62/180,709, 17 Jun. 15,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,455, filed, 12Dec. 14, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708, 24Dec. 14, PROTECTED GUIDE RNAS (PGRNAS); U.S. applications 62/091,462, 12Dec. 14, 62/096,324, 23 Dec. 14, 62/180,681, 17 Jun. 2015, and62/237,496, 5 Oct. 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS;U.S. application 62/091,456, 12 Dec. 14 and 62/180,692, 17 Jun. 2015,ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S.application 62/091,461, 12 Dec. 14, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 14, UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKSAND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING; U.S.application 62/096,761, 24 Dec. 14, ENGINEERING OF SYSTEMS, METHODS ANDOPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S.application 62/098,059, 30 Dec. 14, 62/181,641, 18 Jun. 2015, and62/181,667, 18 Jun. 2015, RNA-TARGETING SYSTEM; U.S. application62/096,656, 24 Dec. 14 and 62/181,151, 17 Jun. 2015, CRISPR HAVING ORASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. application 62/096,697, 24Dec. 14, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application62/098,158, 30 Dec. 14, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETINGSYSTEMS; U.S. application 62/151,052, 22 Apr. 15, CELLULAR TARGETING FOREXTRACELLULAR EXOSOMAL REPORTING; U.S. application 62/054,490, 24 Sep.14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMSAND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLEDELIVERY COMPONENTS; U.S. application 61/939,154, 12 Feb. 14, SYSTEMS,METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,484, 25 Sep. 14,SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,537, 4Dec. 14, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONWITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application62/054,651, 24 Sep. 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. application 62/067,886, 23 Oct.14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMSAND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONSIN VIVO; U.S. applications 62/054,675, 24 Sep. 14 and 62/181,002, 17Jun. 2015, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. application62/054,528, 24 Sep. 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS;U.S. application 62/055,454, 25 Sep. 14, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S.application 62/055,460, 25 Sep. 14, MULTIFUNCTIONAL-CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S.application 62/087,475, 4 Dec. 14 and 62/181,690, 18 Jun. 2015,FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/055,487, 25 Sep. 14, FUNCTIONAL SCREENING WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,546, 4 Dec. 14and 62/181,687, 18 Jun. 2015, MULTIFUNCTIONAL CRISPR COMPLEXES AND/OROPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S.application 62/098,285, 30 Dec. 14, CRISPR MEDIATED IN VIVO MODELING ANDGENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Mention is made of U.S. applications 62/181,659, 18 Jun. 2015 and62/207,318, 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FORSEQUENCE MANIPULATION. Mention is made of U.S. applications 62/181,663,18 Jun. 2015 and 62/245,264, 22 Oct. 2015, NOVEL CRISPR ENZYMES ANDSYSTEMS, U.S. applications 62/181,675, 18 Jun. 2015, 62/285,349, 22 Oct.2015, 62/296,522, 17 Feb. 2016, and 62/320,231, 8 Apr. 2016, NOVELCRISPR ENZYMES AND SYSTEMS, U.S. application 62/232,067, 24 Sep. 2015,U.S. application Ser. No. 14/975,085, 18 Dec. 2015, European applicationNo. 16150428.7, U.S. application 62/205,733, 16 Aug. 2015, U.S.application 62/201,542, 5 Aug. 2015, U.S. application 62/193,507, 16Jul. 2015, and U.S. application 62/181,739, 18 Jun. 2015, each entitledNOVEL CRISPR ENZYMES AND SYSTEMS and of U.S. application 62/245,270, 22Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Mention is also made ofU.S. application 61/939,256, 12 Feb. 2014, and WO 2015/089473(PCT/US2014/070152), 12 Dec. 2014, each entitled ENGINEERING OF SYSTEMS,METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FORSEQUENCE MANIPULATION. Mention is also made of PCT/US2015/045504, 15Aug. 2015, U.S. application 62/180,699, 17 Jun. 2015, and U.S.application 62/038,358, 17 Aug. 2014, each entitled GENOME EDITING USINGCAS9 NICKASES.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

The effectiveness of the present invention has since been demonstrated.Preassembled recombinant CRISPR-Cpf1 complexes comprising Cpf1 and crRNAmay be transfected, for example by electroporation, resulting in highmutation rates and absence of detectable off-target mutations. Hur, J.K. et al, Targeted mutagenesis in mice by electroporation of Cpf1ribonucleoproteins, Nat Biotechnol. 2016 Jun. 6. doi: 10.1038/nbt.3596.[Epub ahead of print]. Genome-wide analyses shows that Cpf1 is highlyspecific. By one measure, in vitro cleavage sites determined for SpCas9in human HEK293T cells were significantly fewer that for SpCas9. Kim, D.et al., Genome-wide analysis reveals specificities of Cpf1 endonucleasesin human cells, Nat Biotechnol. 2016 Jun. 6. doi: 10.1038/nbt.3609.[Epub ahead of print]. An efficient multiplexed system employing Cpf1has been demonstrated in Drosophila employing gRNAs processed from anarray containing inventing tRNAs. Port, F. et al, Expansion of theCRISPR toolbox in an animal with tRNA-flanked Cas9 and Cpf1 gRNAs. doi:http://dx.doi.org/10.1101/046417.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1: Origin and Evolution of Adaptive Immunity Systems

Classification and Annotation of CRISPR-Cas Systems in Archaeal andBacterial Genomes.

The CRISPR-Cas loci has more than 50 gene families and there is nostrictly universal genes, fast evolution, extreme diversity of lociarchitecture. Therefore, no single tree feasible and a multi-prongedapproach is needed. So far, there is comprehensive cas geneidentification of 395 profiles for 93 Cas proteins. Classificationincludes signature gene profiles plus signatures of locus architecture

A new classification of CRISPR-Cas systems is proposed in FIG. 1 . Class2 includes multisubunit crRNA-effector complexes (Cascade) and Class 2includes Single-subunit crRNA-effector complexes (Cas9-like). FIG. 2provides a molecular organization of CRISPR-Cas. FIG. 3 providesstructures of Type I and III effector complexes: commonarchitecture/common ancestry despite extensive sequence divergence. FIG.4 shows CRISPR-Cas as a RNA recognition motif (RRM)-centered system.FIG. 5 shows Cas1 phylogeny where recombination of adaptation andcrRNA-effector modules show a major aspect of CRISPR-Cas evolution. FIG.F shows a CRISPR-Cas census, specifically a distribution of CRISPR-Castypes/subtypes among archaea and bacteria.

Cas1 is not always linked to CRISPR-Cas systems, therefore it may bepossible that there are two branches of “solo” Cas1 which suggests theremay be differences in function and origin and possible novel mobileelements (see Makarova, Krupovic, Koonin, Frontiers Genet 2014). Thegenome organization of three casposon families may provide some clues.In addition to Cas1 and PolB, casposons incorporate diverse genesincluding various nucleases (Krupovic et al. BMC Biology 2014). Onefamily has protein-primed polymerase, another family has RNA-primedpolymerase. In addition to diverse Euryarchaeota and Thaumarchaeota,casposons found in several bacteria which suggests horizontal mobility.Casposon Cas1 (transposase/integrase) suggests a basal clade in the Cas1phylogeny.

Bacteria and archae utilize CRISPR for adaptive immunity in procaryotesand eukaryotes via genome manipulation. Cas 1 provides a ready made toolfor genome manipulation. There are similar mechanisms of integration incasposons and CRISPR, specifically replication-dependent acquisition bycopy/paste not cut-and-paste (Krupovic et al. BMC Biology 2014). Cas1 isa bona fide integrase (Nunez J K, Lee A S, Engelman A, Doudna J A.Integrase-mediated spacer acquisition during CRISPR-Cas adaptiveimmunity. Nature. 2015 Feb. 18). There is similarity between terminalinverted repeats of casposons and CRISPR (Krupovic et al. BMC Biology2014). CRISPR-Cas may have originated from a casposon and an innateimmunity locus (Koonin, Krupovic, Nature Rev Genet, 2015). The evolutionof adaptive immunity systems in prokaryotes and animals may have beenalong parallel courses with transposon integration at innate immunityloci (Koonin, Krupovic, Nature Rev Genet, 2015). RAG1 transposase (thekey enzyme of V(D)J recombination in vertebrates) may have originatedfrom Transib transposons (Kapitonov V V, Jurka J. RAG1 core and V(D)Jrecombination signal sequences were derived from Transib transposons.PLoS Biol. 2005 June; 3(6):e181), however, none of the Transibs encodesRAG2. RAG1 and RAG2 encoding transposons are described in Kapitonov,Koonin, Biol Direct 2015 and Transib transposase phylogeny is presentedin Kapitonov, Koonin, Biol Direct 2015. Defensive DNA elimination inciliates evolved from a PiggyMAc transposon and RNAi, an innate immunesystem (Swart E C, Nowacki M. The eukaryotic way to defend and editgenomes by sRNA-targeted DNA deletion. Ann N Y Acad Sci. 2015).

The relative stability of the classification implies that the mostprevalent variants of CRISPR-Cas systems are already known. However, theexistence of rare, currently unclassifiable variants implies thatadditional types and subtypes remain to be characterized (Makarova etal. 2015. Evolutionary classification of CRISPR-Cas systems and casgenes).

Transposons play a key contribution to the evolution of adaptiveimmunity and other systems involved in DNA manipulation. Class 1CRISPR-Cas originate from transposons but only for an adaptation module.Class 2 CRISPR-Cas have both adaptation and effector functions wheremodules may have evolved from different transposons.

Example 2: New Predicted Class 2 CRISPR-Cas Systems and Evidence oftheir Independent Origins from Transposable Elements

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.These systems are broadly divided into two classes, Class 1 withmultisubunit effector complexes and Class 2 with single-subunit effectormodules exemplified by the Cas9 protein. Applicants developed a simplecomputational pipeline for prediction of putative new Class 2 CRISPR-Cassystems. Analysis of the database of complete bacterial genomes usingthis pipeline resulted in the identification of two new variants, eachrepresented in diverse bacteria and containing cast and cast genes alongwith a third gene encoding a large protein predicted to function as theeffector module. In the first of these loci, the putative effectorprotein (C2c1p) contains a RuvC-like nuclease domain and resembles thepreviously described Cpf1 protein, the predicted effector of Type VCRISPR-Cas systems; accordingly, the new putative system is classifiedas subtype V-B. In depth comparison of protein sequences suggests thatthe RuvC-containing effector proteins, Cas9, Cpf1 and C2C1pindependently evolved from different groups of transposon-encoded TnpBproteins. The second group of new putative CRISPR-Cas loci encompasses alarge protein containing two highly diverged HEPN domains with predictedRNAse activity. Given the novelty of the predicted effector protein,these loci are classified as new Type VI CRISPR-Cas that is likely totarget mRNA. Together, the results of this analysis show that Class2CRISPR-Cas systems evolved on multiple, independent occasions, bycombination of diverse Cas1-Cas2-encoding adaptation modules witheffector proteins derived from different mobile elements. This route ofevolution most likely produced multiple variants of Class 2 systems thatremain to be discovered.

The CRISPR-Cas adaptive immunity systems are present in ˜45% bacterialand ˜90% archaeal genomes and show extreme diversity of Cas proteincomposition and sequence, and genomic loci architecture. Based on thestructural organization of their crRNA-effector complexes, these systemsare divided into two classes, namely class 1, with multisubunit effectorcomplexes, and class 2, with single subunit effector complexes(Makarova, 2015). Class 1 systems are much more common and diverse thanClass 2 systems. Class 1 currently is represented by 12 distinctsubtypes encoded by numerous archaeal and bacterial genomes, whereasclass 2 systems include three subtypes of Type II system and theputative Type V that collectively are found in about 10% of sequencedbacterial genomes (with a single archaeal genome encompassing a putativeType system). Class 2 systems typically contain only three or four genesin the cas operon, namely the cas1-cas2 pair of genes that are involvedin adaptation but not in interference, a single multidomain effectorprotein that is responsible for interference but also contributes to thepre-crRNA processing and adaptation, and often a fourth gene withuncharacterized functions that is dispensable in at least some Type IIsystems. In most cases, a CRISPR array and a gene for a distinct RNAspecies known as tracrRNA (trans-encoded small CRISPR RNA) are adjacentto Class 2 cas operons (Chylinski, 2014). The tracrRNA is partiallyhomologous to the repeats within the respective CRISPR array and isessential for the processing of pre-crRNA that is catalyzed by RNAseIII, a ubiquitous bacterial enzyme that is not associated with theCRISPR-cas loci (Deltcheva, 2011)(Chylinski, 2014; Chylinski, 2013).

The Type II multidomain effector protein Cas9 has been functionally andstructurally characterized in exquisite detail. In different bacteria,Cas9 proteins encompass between about 950 and 1,400 amino acids andcontain two nuclease domains, namely a RuvC-like (RNase H fold) and HNH(McrA-like) nucleases (Makarova, 2011). The crystal structure of Cas9reveals a bilobed organization of the protein, with distinct targetrecognition and nuclease lobes, with the latter accommodating both theRuvC and the HNH domains (Nishimasu, 2014)(Jinek, 2014). Each of thenuclease domains of Cas9 is required for the cleavage of one of thetarget DNA strands (Jinek, 2012; Sapranauskas, 2011). Recently, Cas9 hasbeen shown to contribute to all three stages of the CRISPR response,that is not only target DNA cleavage (interference) but also adaptationand pre-crRNA processing (Jinek, 2012). More specifically, a distinctdomain in the nuclease lobe of Cas9 has been shown to recognize and bindthe Protospacer-Associated Motif (PAM) in viral DNA during theadaptation stage (Nishimasu, 2014)(Jinek, 2014) (Heler, 2015; Wei,2015). At this stage of the CRISPR response, Cas9 forms a complex withCas1 and Cas2, the two proteins that are involved in spacer acquisitionin all CRISPR-Cas systems (Heler, 2015; Wei, 2015).

The Cas9 protein, combined with tracrRNA, has recently become the keytool for the new generation of genome editing and engineering methods(Gasiunas, 2013; Mali, 2013; Sampson, 2014; Cong, 2015). This utility ofCas9 in genome editing hinges on the fact that in Type II CRISPR-Cassystems, unlike other types of CRISPR-Cas systems, all the activitiesrequired for the target DNA recognition and cleavage are assembledwithin a single, albeit large, multidomain protein. This feature of TypeII systems greatly facilitates the design of efficient tools for genomemanipulation. Importantly, not all variants of Cas9 are equal. Most ofthe work so far has been done with Cas9 from Streptococcus pyogenes butother Cas9 species could offer substantial advantages. As a case inpoint, recent experiments with Cas9 from Staphylococcus aureus that isabout 300 amino acids shorter than the S. pyogenes protein have allowedCas9 packaging into the adeno-associated virus vector, resulting in amajor enhancement of CRISPR-Cas utility for genome editing in vivo (Ran,2015).

Type II CRISPR-Cas systems currently are classified into 3 subtypes(II-A, II-B and II-C) (Makarova, 2011)(Fonfara, 2014; Chylinski, 2013;Chylinski, 2014). In addition to the cas1, cas2 and cas9 genes that areshared by all Type II loci, subtype II-A is characterized by an extragene, csn2, that encodes an inactivated ATPase (Nam, 2011; Koo, 2012;Lee, 2012) that plays a still poorly characterized role in spaceracquisition (Barrangou, 2007; Arslan, 2013) (Heler, 2015). Subtype II-Bsystems lack csn2 but instead contains the cas4 gene that is otherwisetypical of Type I systems and encodes a recB family 5′-3′ exonucleasethat contributes to spacer acquisition by generating recombinogeneci DNAends (Zhang, 2012) (Lemak, 2013; Lemak, 2014). The cast and cas2 genesof subtype II-B are most closely related to the respective proteins ofType I CRISPR-Cas systems which implies a recombinant origin of thisType II subtype (Chylinski, 2014).

Subtype II-C CRISPR-Cas systems are the minimal variety that consistsonly of the cas1, cas2 and cas9 genes (Chylinski, 2013; Koonin, 2013;Chylinski, 2014). Notably, however, it has been shown that inCampylobacter jejuni spacer acquisition by the Type II-C systemsrequires the participation of Cas4 encoded by a bacteriophage (Hooton,2014). Another distinct feature of subtype II-C is the formation of someof the crRNAs by transcription involves transcription from internalalternative promoters as opposed to processing observed in all otherexperimentally characterized CRISPR-Cas systems (Zhang, 2013).

Recently, the existence of Type V CRISPR-Cas systems has been predictedby comparative analysis of bacterial genomes. These putative novelCRISPR-Cas systems are represented in several bacterial genomes, inparticular those from the genus Francisella and one archaeon,Methanomethylophilus alvus (Vestergaard, 2014). All putative Type V lociencompass cas1, cas2, a distinct gene denoted cpf1 and a CRISPR array(Schunder, 2013)(Makarova, 2015). Cpf1 is a large protein (about 1300amino acids) that contains a RuvC-like nuclease domain homologous to thecorresponding domain of Cas9 along with a counterpart to thecharacteristic arginine-rich cluster of Cas9. However, Cpf1 lacks theHNH nuclease domain that is present in all Cas9 proteins, and theRuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9where it contains long inserts including the HNH domain (Chylinski,2014; Makarova, 2015). These major differences in the domainarchitectures of Cas9 and Cpf1 suggest that the Cpf1-containing systemsshould be classified as a new type. The composition of the putative TypeV systems implies that Cpf1 is a single-subunit effector complex, andaccordingly, these systems are assigned to Class 2 CRISPR-Cas. Some ofthe putative Type V loci encode Cas4 and accordingly resemble subtypeII-B loci, whereas others lack Cas4 and thus are analogous to subtypeII-C.

It has been shown that the closest homologs of Cas9 and Cpf1 proteinsare TnpB proteins that are encoded in IS605 family transposons andcontain the RuvC-like nuclease domain as well as a Zn-finger that has acounterpart in Cpf1. In addition, homologs of TnpB have been identifiedthat contain a HNH domain inserted into the RuvC-like domain and showhigh sequence similarity to Cas9. The role of TnpB in transposonsremains uncertain as it has been shown that this protein is not requiredfor transposition.

Given the homology of Cas9 and Cpf1 to transposon-encoded proteins,Applicants hypothesized that Class 2 CRISPR-Cas systems could haveevolved on multiple occasions as a result of recombination between atransposon and a cas1-cas2 locus. Accordingly, Applicants devised asimple computational strategy to identify genomic loci that could becandidates for novel variants of Class 2. Here Applicants describe thefirst application of this approach that resulted in the identificationof two groups of such candidates one of which appears to be a distinctsubtype of Type V whereas the second one seems to qualify at Type VI.The new variants of Class2 CRISPR-Cas systems are of obvious interest aspotential tools for genome editing and expression regulation.

Database Search Strategy for Detection of Candidate Novel Class 2CRISPR-Cas Loci.

Applicants implemented a straightforward computational approach toidentify candidate novel Class 2 CRISPR-Cas systems (FIG. 7 . Pipeline).Because the vast majority of the CRISPR-Cas loci encompass a cas1 gene(Makarova, 2011; Makarova, 2015) and the Cas1 sequence is the mosthighly conserved one among all Cas proteins (Takeuchi, 2012), Applicantsreasoned that cas1 is the best possible anchor to identify candidate newloci using translating PSI-BLAST search with Cas1 profiles. Afterdetecting all contigs encoding Cas1, the protein-coding genes werepredicted using GenemarkS within the 20 KB regions upstream anddownstream of the cas1 gene. These predicted genes were annotated usingthe NCBI CDD and Cas protein-specific profiles, and CRISPR arrays werepredicted using the PILER-CR program. This procedure provided forassignment of the detected CRISPR-Cas loci to the known subtypes.Unclassified candidate CRISPR-Cas loci containing large (>500 aa)proteins were selected as candidates for novel Class 2 systems given thecharacteristic presence of such proteins in Types II and V (Cas9 andCpf1, respectively). All 34 candidate loci detected using this criteriawere analyzed on a case by case basis using PSI-BLAST and HHpred. Theprotein sequences encoded in the candidate loci were farther used asqueries to search metagenomic databases for additional homologs, andlong contigs detected in these searches were analyzed as indicatedabove. This analysis pipeline yielded two groups of loci strong links toCRISPR-Cas systems.

Putative Type V-B System.

The first group of candidate loci, provisionally denoted named C2c1(Class 2 candidate 1), is represented in bacterial genomes from fourmajor phyla, including Bacilli, Verrucomicrobia, alpha-proteobacteriaand delta-proteobacteria (FIG. 8 “Organization of complete loci of Class2 systems”). All C2c1 loci encode a Cas1-Cas4 fusion, Cas2, and thelarge protein that Applicants denote C2c1p, and typically, are adjacentto a CRISPR array (FIG. 9 , C2c1 neighborhoods). In the phylogenetictree of Cas1, the respective Cas1 proteins cluster with Type I-U system(FIG. 10 , Cas1 tree), the only one in which the Cas1-Cas4 fusion isfound. The C2c1p proteins consists of approximately 1200 amino acids,and HHpred search detected significant similarity between the C-terminalportion of this protein and a subset of TnpB proteins encoded intransposons of the IS605 family. In contrast, no significant similaritywas detected between C2c1p and Cas9 or Cpf1 that are similar to othergroups of TnpB proteins (Chylinski, 2014)(Makarova, 2015; Makarova,2015). Thus, the domain architecture of C2c1p is similar to that of Cpf1and distinct from that of Cas9 although all three Cas proteins seem tohave evolved from the TnpB family (FIG. 11 “Domain organization of class2 families”). The N-terminal region of C2c1p shows no significantsimilarity to other proteins. Secondary structure prediction indicatesthat this region adopts mostly alpha-helical conformation. The twosegments of similarity with TnpB encompass the three catalytic motifs ofthe RuvC-like nuclease, with the D..E..D signature (FIG. 12 , “TnpBhomology regions in Class 2 proteins”); the region corresponding to thebridge helix (also known as arginine-rich cluster) that in Cas9 proteinis involved in crRNA-binding; and a small region that appears to be thecounterpart to the Zn finger of TnpB (however, the Zn-binding cysteineresidues are replaced in C2C1p indicating that this protein does notbind zinc). The similarity of the domain architectures of C2c1p and Cpf1suggests that the C2c1 loci are best classified as Subtype V-B in whichcase the Cpf1-encoding loci become Subtype V-A.

Despite similarity of cas1 genes associated with this system, the CRISPRrepeats in the respective arrays are highly heterogeneous although allof them are 36-37 bp long and can be classified as unstructured (foldingenergy, ΔG, is −0.5-4.5 kcal/mole whereas highly palindromic CRISPR haveΔG below −7). According to the CRISPRmap (Lange, 2013) classificationscheme, several of the Subtype V-B repeats share some sequence orstructural similarity with Type II repeats.

Considering the possibility that the putative Subtype V-B CRISPR-Cassystems are mechanistically analogous to Type II systems, Applicantsattempted to identify the tracrRNA in the respective genomic loci

Comparison of the spacers from the Type V-B CRISPR arrays to thenon-redundant nucleotide sequence database identified several matches tovarious bacterial genomes. The relevance of these matches is difficultto assess, considering that no phages are known for the bacteria thatharbor putative Type V-B CRISPR-Cas systems.

Putative Type VI Systems.

The second group of candidate CRISPR-Cas loci, denoted C2c2, wasidentified in genomes from 5 major bacterial phyla,alpha-proteobacteria, Bacilli, Clostridia, Fusobacteria andBacteroidetes (FIG. 8 “Organization of complete loci of Class 2systems”). Similar to c2c1, the C2c2 loci encompass cas1 and cas2 genesalong with a large protein (C2c2p) and a CRISPR array; however, unlikeC2c1, C2c2p is often encoded next to a CRISPR array but not cas1-cas2(FIG. 13 , C2c2 neighborhoods). In the phylogenetic tree of Cas1, theCas1 proteins from the C2c2 loci are distributed among two clades. Thefirst clade includes Cas1 from Clostridia and is located within the TypeII subtree along with a small Type III-A branch (FIG. 10 , Cas1 tree).The second clade consists of Cas1 proteins from C2c2 loci ofLeptotrichia and is lodged inside a mixed branch that mostly containsCas1 proteins from Type III-A CRISPR-Cas systems. Database searchesusing HHpred and PSI-BLAST detected no sequence similarity between C2c2pand other proteins. However, inspection of multiple alignments of C2c2pprotein sequences led to the identification of two strictly conservedRxxxxH motifs that are characteristic of HEPN domains (Anantharaman,2013). Secondary structure predictions indicates that these motifs arelocated within structural contexts compatible with the HEPN domainstructure as is the overall secondary structure prediction for therespective portions of C2c2p. The HEPN domains are small (˜150 aa) alphahelical domains that have been shown or predicted to possess RNAseactivity and are often associated with various defense systems(Anantharaman, 2013) (FIG. 14 , HEPN RxxxxH motif in C2c2 family). Thesequences of HEPN domains show little conservation except for thecatalytic RxxxxH motif. Thus, it appears likely that C2c2p contains twoactive HEPN domains. The HEPN domain is not new to CRISPR-Cas systems asit is often associated with the CARF (CRISPR-Associated Rossmann Fold)domain in Csm6 and Csx1 proteins that are present in many Type IIICRISPR-Cas systems (Makarova, 2014). These proteins do not belong toeither the adaptation modules or effector complexes but rather appear tobe components of the associated immunity module that is present in themajority of CRISPR-Cas systems and is implicated in programmed celldeath as well as regulatory functions during the CRISPR response(Koonin, 2013; Makarova, 2012; Makarova, 2013). However, C2c2p differsfrom Csm6 and Csx1 in that this much larger protein is the only oneencoded in the C2c2 loci, except for Cas1 and Cas2. Thus, it appearslikely that C2c2p is the effector of these putative novel CRISPR-Cassystems and the HEPN domains are the catalytic moieties thereof. Outsideof the predicted HEPN domains, the C2c1p sequence showed no detectablesimilarity to other proteins and is predicted to adopt a mixedalpha/beta secondary structure.

The CRISPR arrays in the C2c2 loci are highly heterogeneous, with thelength of 35 to 39 bp, and unstructured (folding energy of −0.9 to 4.7kcal/mole). According to CRISPRmap (Lange, 2013), these CRISPR do notbelong to any of the established structural classes and are assigned to3 of the 6 superclasses. Only the CRISPR from Listeria seeligeri wasassigned to the sequence family 24 that is usually associated with TypeII-C systems.

Spacer analysis of the C2c2 loci identified one 30 nucleotide regionidentical to a genomic sequence from Listeria weihenstephanensis and twoimperfect hits to bacteriophage genomes.

Given the unique predicted effector complex of C2c2, these systems seemto qualify as a putative Type VI CRISPR-Cas. Furthermore, taking intoaccount that all experimentally characterized and enzymatically activeHEPN domains are RNAses, Type VI systems are likely to act at the levelof mRNA.

Applicants applied a simple, straightforward computational strategy topredict new Class 2 CRISPR-cas systems. The previously described class 2systems, namely Type II and the putative Type V, consisted of the cas1and cas2 genes (and in some cases also cas4) comprising the adaptationmodule and a single large protein that comprises the effector module.Therefore, Applicants surmised that any genomic locus containing cas1and a large protein could be a potential candidate for a novel Class 2system that merits detailed investigation. Such analysis using sensitivemethods for protein sequence comparison led to the identification of twostrong candidates one of which is a subtype of the previously describedputative Type V whereas the other one qualifies as a new putative TypeVI, on the strength of the presence of a presence of a novel predictedeffector protein. Many of these new systems occur in bacterial genomesthat encompass no other CRISPR-Cas loci suggesting that Type V and TypeVI systems can function autonomously.

Combined with the results of previous analyses, (Chylinski, 2014;Makarova, 2011), the identification of the putative Type V-B reveals thedominant theme in the evolution of Class 2 CRISPR-Cas systems. Theeffector proteins of all currently known systems of this class appear tohave evolved from the pool of transposable elements that encode TnpBproteins containing the RuvC-like domain. The sequences of the RuvC-likedomains of TnpB and the homologous domains of the Class 2 effectorproteins are too diverged for reliable phylogenetic analysis.Nevertheless, for Cas9, the effector protein of Type II systems, thespecific ancestor seems to be readily identifiable, namely a family ofTnpB-like proteins, particularly abundant in Cyanobacteria, that show arelatively high sequence similarity to Cas9 and share with it the entiredomain architecture, namely the RuvC-like and HNH nuclease domains andthe arginine-rich bridge helix (Chylinski, 2014) (FIG. 11 , “Domainorganization of class 2 families”; FIG. 12 , “TnpB homology regions inClass 2 proteins”). Unlike Cas9, it was impossible to trace Cpf1 andC2c1 to a specific TnpB family; despite the conservation of all motifscentered at the catalytic residues of the RuvC-like nucleases, theseproteins show only a limited similarity to generic profiles of the TnpB.However, given that C2c1p shows no detectable sequence similarity withCpf1, contains distinct insertions between the RuvC-motifs and clearlyunrelated N-terminal regions, it appears most likely that Cpf1 and C2c1originated independently from different families within the pool ofTnpB-encoding elements.

It is intriguing that the TnpB proteins seem to be “predesigned” forutilization in Class 2 CRISPR-Cas effector complexes such that theyapparently have been recruited on multiple different occasions.Conceivably, such utility of TnpB proteins has to do with theirpredicted ability to cut a single-stranded DNA while bound to a RNAmolecule via the R-rich bridge helix that in Cas9 has been shown to bindcrRNA (Jinek, 2014; Nishimasu, 2014). The functions of TnpB are poorlyunderstood. This protein is not required for transposition, and in onecase, has been shown to down-regulate transposition (Pasternak, 2013)but their mechanism of action remains unknown. Experimental study ofTnpB is likely to shed light on the mechanistic aspects of the Class 2CRISPR-Cas systems. It should be noted that the mechanisms of Cpf1 andC2c1 could be similar to each other but are bound to substantiallydiffer from that of Cas9 because the former two proteins lack the HNHdomain that in Cas9 is responsible for nicking one of the target DNAstrands (Gasiunas, 2012) (Jinek, 2012) (Chen, 2014). Accordingly,exploitation of Cpf1 and C2c1 might bring additional genome editingpossibilities.

In evolutionary terms, it is striking that Class 2 CRISPR-Cas appear tobe completely derived from different transposable elements given therecent evidence on the likely origin of cas1 genes from a distincttransposon family (Koonin, 2015; Krupovic, 2014). Furthermore, thelikely independent origin of the effector proteins from differentfamilies of TnpB, along with the different phylogenetic affinities ofthe respective cas1 proteins, strongly suggest that Class 2 systems haveevolved on multiple occasions through the combination of variousadaptation modules and transposon-derived nucleases giving rise toeffector proteins. This mode of evolution appears to be the ultimatemanifestation of the modularity that is characteristic of CRISPR-Casevolution (Makarova, 2015), with the implication that additionalcombinations of adaptation and effector module are likely to exist innature.

The putative Type VI CRISPR-Cas systems encompass a predicted noveleffector protein that contains two predicted HEPN domain that are likelyto possess RNAse activity. The HEPN domains are not parts of theeffector complexes in other CRISPR-Cas systems but are involved in avariety of defense functions including a predicted ancillary role invarious CRISPR-Cas systems (Anantharaman, 2013) (Makarova, 2015). Thepresence of the HEPN domains as the catalytic moiety of the predictedeffector module implies that the Type VI systems target and cleave mRNA.Previously, mRNA targeting has been reported for certain Type IIICRISPR-Cas systems (Hale, 2014; Hale, 2009) (Peng, 2015). Although HEPNdomains so far have not been detected in bona fide transposableelements, they are characterized by high horizontal mobility and areintegral to mobile elements such as toxin-antitoxin units (Anantharaman,2013). Thus, the putative Type VI systems seem to fit the generalparadigm of the modular evolution of Class 2 CRISPR-Cas from mobilecomponents, and additional variants and new types are expected to bediscovered by analysis of genomic and metagenomics data.

Modular evolution is a key feature of CRISPR-Cas systems. This mode ofevolution appears to be most pronounced in Class 2 systems that evolvethrough the combination of adaptation modules from various otherCRISPR-Cas systems with effector proteins that seem to be recruited frommobile elements on multiple independent occasions. Given the extremediversity of mobile elements in bacteria, it appears likely thateffector modules of Class 2 CRISPR-Cas systems are highly diverse aswell. Here Applicants employed a simple computational approach todelineate two new variants of CRISPR-Cas systems but many more arelikely to exist bacterial genomes that have not yet been sequenced.Although most if not all of these new CRISPR-Cas systems are expected tobe rare, they could employ novel strategies and molecular mechanisms andwould provide a major resource for new applications in genomeengineering and biotechnology.

TBLASTN program was used to search with Cas1 profile as a query againstNCBI WGS database. Sequences of contigs or complete genome partitionswhere Cas1 hit has been identified where retrieved from the samedatabase. The region around the Cas1 gene was cut out and translatedusing GENMARK. Predicted proteins for each were searched against acollection of profiles from CDD database (Marchler-Bauer, 2009) andspecific Cas profiles available at FTP, with hit priority to Casproteins. Procedure to identify completeness of CRISPR loci developedpreviously has been applied to each locus.

CRISPRmap (Lange, 2013) was used for repeat classification.

Iterative profile searches with the PSI-BLAST (Altschul, 1997) andcomposition based-statistics and low complexity filtering turned off,were used to search for distantly similar sequences both NCBI'snon-redundant (NR) database. Each identified non-redundant protein wassearched against WGS using TBLAST program. HHpred was used with defaultparameters was used to identify remote sequence similarity (Soding,2005). Multiple sequence alignments were constructed using MUSCLE(Edgar, 2004). Protein secondary structure was predicted using Jpred 4(Drozdetskiy, 2015).

Chosen Gene Candidates

Gene ID: A; Gene Type: C2C1; Organism: 5. Opitutaceae bacterium TAV5; SpacerLength - mode (range): 34 (33 to 37);  DR1: (SEQ ID NO: 27)GCCGCAGCGAAUGCCGUUUCACGAAUCGUCAGGCGG; DR2: none; tracrRNA1:(SEQ ID NO: 28) GCUGGAGACGUUUUUUGAAACGGCGAGUGCUGCGGAUAGCGAGUUUCUCUUGGGGAGGCGCUCGCGGCCACUUUU; tracrRNA2: none; Protein Sequence:(SEQ ID NO: 29) MSLNRIYQGRVAAVETGTALAKGNVEWMPAAGGDEVLWQHHELFQAAINYYLVALLALADKNNPVLGPLISQMDNPQSPYHVWGSFRRQGRQRTGLSQAVAPYITPGNNAPTLDEVFRSILAGNPTDRATLDAALMQLLKACDGAGAIQQEGRSYWPKFCDPDSTANFAGDPAMLRREQHRLLLPQVLHDPAITIADSPALGSFDTYSIATPDTRTPQLTGPKARARLEQAITLWRVRLPESAADFDRLASSLKKIPDDDSRLNLQGYVGSSAKGEVQARLFALLLFRHLERSSFTLGLLRSATPPPKNAETPPPAGVPLPAASAADPVRIARGKRSFVFRAFTSLPCWHGGDNIHPTWKSFDIAAFKYALTVINQIEEKTKERQKECAELETDFDYMHGRLAKIPVKYTTGEAEPPPILANDLRIPLLRELLQNIKVDTALTDGEAVSYGLQRRTIRGFRELRRIWRGHAPAGTVFSSELKEKLAGELRQFQTDNSTTIGSVQLFNELIQNPKYWPIWQAPDVETARQWADAGFADDPLAALVQEAELQEDIDALKAPVKLTPADPEYSRRQYDFNAVSKFGAGSRSANRHEPGQTERGHNTFTTEIAARNAADGNRWRATHVRIHYSAPRLLRDGLRRPDTDGNEALEAVPWLQPMMEALAPLPTLPQDLTGMPVFLMPDVTLSGERRILLNLPVTLEPAALVEQLGNAGRWQNQFFGSREDPFALRWPADGAVKTAKGKTHIPWHQDRDHFTVLGVDLGTRDAGALALLNVTAQKPAKPVHRIIGEADGRTWYASLADARMIRLPGEDARLFVRGKLVQEPYGERGRNASLLEWEDARNIILRLGQNPDELLGADPRRHSYPEINDKLLVALRRAQARLARLQNRSWRLRDLAESDKALDEIHAERAGEKPSPLPPLARDDAIKSTDEALLSQRDIIRRSFVQIANLILPLRGRRWEWRPHVEVPDCHILAQSDPGTDDTKRLVAGQRGISHERIEQIEELRRRCQSLNRALRHKPGERPVLGRPAKGEEIADPCPALLEKINRLRDQRVDQTAHAILAAALGVRLRAPSKDRAERRHRDIHGEYERFRAPADFVVIENLSRYLSSQDRARSENTRLMQWCHRQIVQKLRQLCETYGIPVLAVPAAYSSRFSSRDGSAGFRAVHLTPDHRHRMPWSRILARLKAHEEDGKRLEKTVLDEARAVRGLFDRLDRFNAGHVPGKPWRTLLAPLPGGPVFVPLGDATPMQADLNAAINIALRGIAAPDRHDIHHRLRAENKKRILSLRLGTQREKARWPGGAPAVTLSTPNNGASPEDSDALPERVSNLFVDIAGVANFERVTIEGVSQKFATGRGLWASVKQRAWNRVARLNETVTDNNRNEEEDDIPMGene ID: B; Gene Type: C2C1; Organism: 7. Bacillus thermoamylovorans strainB4166; Spacer Length - mode (range): 37 (35-38); DR1: (SEQ ID NO: 30)GUCCAAGAAAAAAGAAAUGAUACGAGGCAUUAGCAC; DR2: none; tracrRNA1:(SEQ ID NO: 31) CUGGACGAUGUCUCUUUUAUUUCUUUUUUCUUGGAUCUGAGUACGAGCACCCACAUUGGACAUUUCGCAUGGUGGGUGCUCGUACUAUAGGUAAAACAAACCUUUUU; tracrRNA2: none;Protein Sequence: (SEQ ID NO: 32)MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDVVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPFTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEHKTLEERIKEDIQAFKSLEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKFVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKLVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWGNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMGene ID: C; Gene Type: C2C1; Organism: 9. Bacillus sp. NSP2.1; Spacer Length -mode (range): 36(35-42); DR1: (SEQ ID NO: 33)GUUCGAAAGCUUAGUGGAAAGCUUCGUGGUUAGCAC; DR2: none; tracrRNA1:(SEQ ID NO: 34) CACGGAUAAUCACGACUUUCCACUAAGCUUUCGAAUUUUAUGAUGCGAGCAUCCUCUCAGGUCAAAAAA; tracrRNA2: none; Protein Sequence: (SEQ ID NO: 35)MAIRSIKLKLKTHTGPEAQNLRKGIWRTHRLLNEGVAYYMKMLLLFRQESTGERPKEELQEELICHIREQQQRNQADKNTQALPLDKALEALRQLYELLVPSSVGQSGDAQIISRKFLSPLVDPNSEGGKGTSKAGAKPTWQKKKEANDPTWEQDYEKWKKRREEDPTASVITTLEEYGIRPIFPLYTNTVTDIAWLPLQSNQFVRTWDRDMLQQAIERLLSWESWNKRVQEEYAKLKEKMAQLNEQLEGGQEWISLLEQYEENRERELRENMTAANDKYRITKRQMKGWNELYELWSTFPASASHEQYKEALKRVQQRLRGRFGDAHFFQYLMEEKNRLIWKGNPQRIHYFVARNELTKRLEEAKQSATMTLPNARKHPLWVRFDARGGNLQDYYLTAEADKPRSRRFVTFSQLIWPSESGWMEKKDVEVELALSRQFYQQVKLLKNDKGKQKIEFKDKGSGSTFNGHLGGAKLQLERGDLEKEEKNFEDGEIGSVYLNVVIDFEPLQEVKNGRVQAPYGQVLQLIRRPNEFPKVTTYKSEQLVEWIKASPQHSAGVESLASGFRVMSIDLGLRAAAATSIFSVEESSDKNAADFSYWIEGTPLVAVHQRSYMLRLPGEQVEKQVMEKRDERFQLHQRVKFQIRVLAQIMRMANKQYGDRWDELDSLKQAVEQKKSPLDQTDRTFWEGIVCDLTKVLPRNEADWEQAVVQIHRKAEEYVGKAVQAWRKRFAADERKGIAGLSMWNIEELEGLRKLLISWSRRTRNPQEVNRFERGHTSHQRLLTHIQNVKEDRLKQLSHAIVMTALGYVYDERKQEWCAEYPACQVILFENLSQYRSNLDRSTKENSTLMKWAHRSIPKYVHMQAEPYGIQIGDVRAEYSSRFYAKTGTPGIRCKKVRGQDLQGRRFENLQKRLVNEQFLTEEQVKQLRPGDIVPDDSGELFMTLTDGSGSKEVVFLQADINAAHNLQKRFWQRYNELFKVSCRVIVRDEEEYLVPKTKSVQAKLGKGLFVKKSDTAWKDVYVWDSQAKLKGKTTFTEESESPEQLEDFQEIIEEAEEAKGTYRTLFRDPSGVFFPESVWYPQKDFWGEVKRKLYGKLRERFLTKARGene ID: D; Gene Type: C2C2; Organism: 4. Lachnospiraceae bacterium NK4A144G619; Spacer Length - mode (range): 35; DR1: (SEQ ID NO: 36)GUUUUGAGAAUAGCCCGACAUAGAGGGCAAUAGAC; DR2: (SEQ ID NO: 37)GUUAUGAAAACAGCCCGACAUAGAGGGCAAUAGACA; tracrRNA1: none; tracrRNA2: none;Protein Sequence: (SEQ ID NO: 38)MKISKVDHTRMAVAKGNQHRRDEISGILYKDPTKTGSIDFDERFKKLNCSAKILYHVFNGIAEGSNKYKNIVDKVNNNLDRVLFTGKSYDRKSIIDIDTVLRNVEKINAFDRISTEEREQIIDDLLEIQLRKGLRKGKAGLREVLLIGAGVIVRTDKKQEIADFLE1LDEDFNKTNQAKNIKLSIENQGLVVSPVSRGEERIFDVSGAQKGKSSKKAQEKEALSAFLLDYADLDKNVRFEYLRKIRRLINLYFYVKNDDVMSLTEIPAEVNLEKDFDIWRDHEQRKEENGDFVGCPDILLADRDVKKSNSKQVKIAERQLRESIREKNIKRYRFSIKTIEKDDGTYFFANKQISVFWIHRIENAVERILGSINDKKLYRLRLGYLGEKVWKDILNFLSIKYIAVGKAVFNFAMDDLQEKDRDIEPGKISENAVNGLTSFDYEQIKADEMLQREVAVNVAFAANNLARVTVDIPQNGEKEDILLWNKSDIKKYKKNSKKGILKSILQFFGGASTWNMKMFEIAYHDQPGDYEENYLYDIIQIIYSLRNKSFHFKTYDHGDKNWNRELIGKMIEHDAERVISVEREKFHSNNLPMFYKDADLKKILDLLYSDYAGRASQVPAFNTVLVRKNFPEFLRKDMGYKVHFNNPEVENQWHSAVYYLYKEIYYNLFLRDKEVKNLFYTSLKNIRSEVSDKKQKLASDDFASRCEEIEDRSLPEICQIIMTEYNAQNFGNRKVKSQRVIEKNKDIFRHYKMLLIKTLAGAFSLYLKQERFAFIGKATPIPYETTDVKNFLPEWKSGMYASFVEEIKNNLDLQEWYIVGRFLNGRMLNQLAGSLRSYIQYAEDIERRAAENRNKLFSKPDEKIEACKKAVRVLDLCIKISTRISAEFTDYFDSEDDYADYLEKYLKYQDDAIKELSGSSYAALDHFCNKDDLKFDIYVNAGQKPILQRNIVMAKLFGPDNILSEVMEKVTESAIREYYDYLKKVSGYRVRGKCSTEKEQEDLLKFQRLKNAVEFRDVTEYAEVINELLGQLISWSYLRERDLLYFQLGFHYMCLKNKSFKPAEYVDIRRNNGTIIHNAILYQIVSMYINGLDFYSCDKEGKTLKPIETGKGVGSKIGQFIKYSQYLYNDPSYKLEIYNAGLEVFENIDEHDNITDLRKYVDHFKYYAYGNKMSLLDLYSEFFDRFFTYDMKYQKNVVNVLENILLRHFVIFYPKFGSGKKDVGIRDCKKERAQIEISEQSLTSEDFMFKLDDKAGEEAKKFPARDERYLQTIAKLLYYPNEIEDMNRFMKKGETINKKVQFNRKKKITRKQKNNSSNEVLSSTMGYLFKNIKLGene ID: E; Gene Type: C2C2; Organism: 8. Listeria seeligeri serovar 1/2b str.SLCC3954; Spacer Length - mode (range): 30; DR1: (SEQ ID NO: 39)GUUUUAGUCCUCUUUCAUAUAGAGGUAGUCUCUUAC; DR2: none; tracrRNA1 :(SEQ ID NO: 40) AUGAAAAGAGGACUAAAACUGAAAGAGGACUAAAACACCAGAUGUGGAUAACUAUAUUAGUGGCUAUUAAAAAUUCGUCGAUAUUAGAGAGGAAACUUU; tracrRNA2: none;Protein Sequence: (SEQ ID NO: 41)MWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKVLISRDKNGGKLVYENEMQDNTEQIMHHKKSSFYKSVVNKTICRPEQKQMKKLVHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMKGene ID: F; Gene Type: C2C2; Organism: 12. Leptotrichia wadei F0279; SpacerLength - mode (range): 31; DR1: (SEQ ID NO: 42)GUUUUAGUCCCCUUCGUUUUUGGGGUAGUCUAAAUC; DR2: none; tracrRNA1:(SEQ ID NO: 43) GAUUUAGAGCACCCCAAAAGUAAUGAAAAUUUGCAAUUAAAUAAGGAAUAUUAAAAAAAUGUGAUUUUAAAAAAAUUGAAGAAAUUAAAUGAAAAAUUGUCCAAGUAA AAAAA; tracrRNA2:(SEQ ID NO: 44) AUUUAGAUUACCCCUUUAAUUUAUUUUACCAUAUUUUUCUCAUAAUGCAAACUAAUAUUCCAAAAUUUUU; Protein Sequence: (SEQ ID NO: 45)MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTLGene ID: G; Gene Type: C2C2; Organism: 14. Leptotrichia shahii DSM 19757 B031;Spacer Length - mode (range): 30 (30-32); DR1: (SEQ ID NO: 46)GUUUUAGUCCCCUUCGAUAUUGGGGUGGUCUAUAUC; DR2: none; tracrRNA1:(SEQ ID NO: 47) AUUGAUGUGGUAUACUAAAAAUGGAAAAUUGUAUUUUUGAUUAGAAAGAUGUAAAAUUGAUUUAAUUUAAAAAUAUUUUAUUAGAUUAAAGUAGA; tracrRNA2: none;Protein Sequence: (SEQ ID NO: 48)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNMARDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERIALAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNGene ID: H; Gene Type: Cpf1; Organism: Francisella ularensis sub sp. novicidaU112; Spacer Length - mode (range): 31; DR1: (SEQ ID NO: 49)GUCUAAGAACUUUAAAUAAUUUCUACUGUUGUAGAU;; DR2: none; tracrRNA1:(SEQ ID NO: 50) AUCUACAAAAUUAUAAACUAAAUAAAGAUUCUUAUAAUAACUUUAUAUAUAAUCGAAAUGUAGAGAAUUUU; tracrRNA2: none; Protein Sequence: (SEQ ID NO: 51)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKEEPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVREIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Genes for Synthesis

For genes A through H, optimize for human expression and append thefollowing DNA sequence to the end of each gene. Note this DNA sequencecontains a stop codon (underlined), so do not add any stop codon to thecodon optimized gene sequence:

(SEQ ID NO: 52) AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGggatccTACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAA

For optimization, avoid the following restriction sites: BamHI, EcoRI,HindIII, BsmBI, BsaI, BbsI, AgeI, XhoI, NdeI, NotI, KpnI, BsrGI, SpeI,Xba1, NheI

These genes are cloned into a simple mammalian expression vector:

>A (SEQ ID NO: 53)MSLNRIYQGRVAAVETGTALAKGNVEWMPAAGGDEVLWQHHELFQAAINYYLVALLALADKNNPVLGPLISQMDNPQSPYHVWGSFRRQGRQRTGLSQAVAPYITPGNNAPTLDEVFRSILAGNPTDRATLDAALMQLLKACDGAGAIQQEGRSYWPKFCDPDSTANFAGDPAMLRREQHRLLLPQVLHDPAITHDSPALGSFDTYSIATPDTRTPQLTGPKARARLEQAITLWRVRLPESAADFDRLASSLKKIPDDDSRLNLQGYVGSSAKGEVQARLFALLLFRHLERSSFTLGLLRSATPPPKNAETPPPAGVPLPAASAADPVRIARGKRSFVFRAFTSLPCWHGGDNIRPTWKSFDIAAFKYALTVINQIEEKTKERQKECAELETDFDYMHGRLAKIPVKYTTGEAEPPPILANDLRIPLLRELLQNIKVDTALTDGEAVSYGLQRRTIRGFRELRRIWRGHAPAGTVFSSELKEKLAGELRQFQTDNSTTIGSVQLFNELIQNPKYWPIWQAPDVETARQWADAGFADDPLAALVQEAELQEDIDALKAPVKLTPADPEYSRRQYDFNAVSKFGAGSRSANRHEPGQTERGHNTFTTEIAARNAADGNRWRATHVRIHYSAPRLLRDGLRRPDTDGNEALEAVPWLQPMMEALAPLPTLPQDLTGMPVFLMPDVTLSGERRILLNLPVTLEPAALVEQLGNAGRWQNQFFGSREDPFALRWPADGAVKTAKGKTHIPWHQDRDEIFTVLGVDLGTRDAGALALLNVTAQKPAKPVHRIIGEADGRTWYASLADARMIRLPGEDARLFVRGKLVQEPYGERGRNASLLEWEDARNIILRLGQNPDELLGADPRRHSYPEINDKLLVALRRAQARLARLQNRSWRLRDLAESDKALDEIHAERAGEKPSPLPPLARDDAIKSTDEALLSQRDIIRRSFVQIANLILPLRGRRWEWRPHVEVPDCHILAQSDPGTDDTKRLVAGQRGISHERIEQIEELRRRCQSLNRALRHKPGERPVLGRPAKGEEIADPCPALLEKINRLRDQRVDQTAHAILAAALGVRLRAPSKDRAERRHRDIHGEYERFRAPADFVVIENLSRYLSSQDRARSENTRLMQWCHRQIVQKLRQLCETYGIPVLAVPAAYSSRFSSRDGSAGFRAVHLTPDHRHRMPWSRILARLKAHEEDGKRLEKTVLDEARAVRGLFDRLDRFNAGHVPGKPWRTLLAPLPGGPVFVPLGDATPMQADLNAAINIALRGIAAPDRHDIHHRLRAENKKRILSLRLGTQREKARWPGGAPAVTLSTPNNGASPEDSDALPERVSNLFVDIAGVANFERVTIEGVSQKFATGRGLWASVKQRAWNRVARLNETVTDNNRNEEEDDIPM >B (SEQ ID NO: 54)MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDVVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPFTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEHKTLEERIKEDIQAFKSLEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKFVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKLVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWGNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSM >C (SEQ ID NO: 55)MAIRSIKLKLKTHTGPEAQNLRKGIWRTHRLLNEGVAYYMKMLLLFRQESTGERPKEELQEELICHIREQQQRNQADKNTQALPLDKALEALRQLYELLVPSSVGQSGDAQIISRKFLSPLVDPNSEGGKGTSKAGAKPTWQKKKEANDPTWEQDYEKWKKRREEDPTASVITTLEEYGIRPIFPLYTNTVTDIAWLPLQSNQFVRTWDRDMLQQAIERLLSWESWNKRVQEEYAKLKEKMAQLNEQLEGGQEWISLLEQYEENRERELRENMTAANDKYRITKRQMKGWNELYELWSTFPASASHEQYKEALKRVQQRLRGRFGDAHFFQYLMEEKNRLIWKGNPQRIHYFVARNELTKRLEEAKQSATMTLPNARKHPLWVRFDARGGNLQDYYLTAEADKPRSRRFVTFSQLIWPSESGWMEKKDVEVELALSRQFYQQVKLLKNDKGKQKIEFKDKGSGSTFNGHLGGAKLQLERGDLEKEEKNFEDGEIGSVYLNVVIDFEPLQEVKNGRVQAPYGQVLQLIRRPNEFPKVTTYKSEQLVEWIKASPQHSAGVESLASGFRVMSIDLGLRAAAATSIFSVEESSDKNAADFSYWIEGTPLVAVHQRSYMLRLPGEQVEKQVMEKRDERFQLHQRVKFQIRVLAQIMRMANKQYGDRWDELDSLKQAVEQKKSPLDQTDRTFWEGIVCDLTKVLPRNEADWEQAVVQIHRKAEEYVGKAVQAWRKRFAADERKGIAGLSMWNIEELEGLRKLLISWSRRTRNPQEVNRFERGHTSHQRLLTHIQNVKEDRLKQLSHAIVMTALGYVYDERKQEWCAEYPACQVILFENLSQYRSNLDRSTKENSTLMKWAHRSIPKYVHMQAEPYGIQIGDVRAEYSSRFYAKTGTPGIRCKKVRGQDLQGRRFENLQKRLVNEQFLTEEQVKQLRPGDIVPDDSGELFMTLTDGSGSKEVVFLQADINAAHNLQKRFWQRYNELFKVSCRVIVRDEEEYLVPKTKSVQAKLGKGLFVKKSDTAWKDVYVWDSQAKLKGKTTFTEESESPEQLEDFQEIIEEAEEAKGTYRTLFRDPSGVFFPESVWYPQKDFWGEVKRKLYGKLRERF LTKAR >D(SEQ ID NO: 56) MKISKVDHTRMAVAKGNQHRRDEISGILYKDPTKTGSIDFDERFKKLNCSAKILYHVFNGIAEGSNKYKNIVDKVNNNLDRVLFTGKSYDRKSIIDIDTVLRNVEKINAFDRISTEEREQIIDDLLEIQLRKGLRKGKAGLREVLLIGAGVIVRTDKKQEIADFLEILDEDFNKTNQAKNIKLSIENQGLVVSPVSRGEERIFDVSGAQKGKSSKKAQEKEALSAFLLDYADLDKNVRFEYLRKIRRLINLYFYVKNDDVMSLTEIPAEVNLEKDFDIWRDHEQRKEENGDFVGCPDILLADRDVKKSNSKQVKIAERQLRESIREKNIKRYRFSIKTIEKDDGTYFFANKQISVFWIHRIENAVERILGSINDKKLYRLRLGYLGEKVWKDILNFLSIKYIAVGKAVFNFAMDDLQEKDRDIEPGKISENAVNGLTSFDYEQIKADEMLQREVAVNVAFAANNLARVTVDIPQNGEKEDILLWNKSDIKKYKKNSKKGILKSILQFFGGASTWNMKMFEIAYHDQPGDYEENYLYDIIQIIYSLRNKSFHFKTYDHGDKNWNRELIGKMIEHDAERVISVEREKFHSNNLPMFYKDADLKKILDLLYSDYAGRASQVPAFNTVLVRKNFPEFLRKDMGYKVHFNNPEVENQWHSAVYYLYKEIYYNLFLRDKEVKNLFYTSLKNIRSEVSDKKQKLASDDFASRCEEIEDRSLPEICQIIMTEYNAQNFGNRKVKSQRVIEKNKDIFRHYKMLLIKTLAGAFSLYLKQERFAFIGKATPIPYETTDVKNFLPEWKSGMYASFVEEIKNNLDLQEWYIVGRFLNGRMLNQLAGSLRSYIQYAEDIERRAAENRNKLFSKPDEKIEACKKAVRVLDLCIKISTRISAEFTDYFDSEDDYADYLEKYLKYQDDAIKELSGSSYAALDHFCNKDDLKFDIYVNAGQKPILQRNIVMAKLFGPDNILSEVMEKVTESAIREYYDYLKKVSGYRVRGKCSTEKEQEDLLKFQRLKNAVEFRDVTEYAEVINELLGQLISWSYLRERDLLYFQLGFHYMCLKNKSFKPAEYVDIRRNNGTIIHNAILYQIVSMYINGLDFYSCDKEGKTLKPIETGKGVGSKIGQFIKYSQYLYNDPSYKLEIYNAGLEVFENIDEHDNITDLRKYVDHFKYYAYGNKMSLLDLYSEFFDRFFTYDMKYQKNVVNVLENILLRHFVIFYPKFGSGKKDVGIRDCKKERAQIEISEQSLTSEDFMFKLDDKAGEEAKKFPARDERYLQTIAKLLYYPNEIEDMNRFMKKGETINKKVQFNRKKKITRKQKNNSSNEVLSSTMGYLFKNIKL >E (SEQ ID NO: 57)MWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKVLISRDKNGGKLVYENEMQDNTEQIMHHKKSSFYKSVVNKTICRPEQKQMKKLVHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHALKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIELPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMK >F (SEQ ID NO: 58)MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVELEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPREINEFVREIKLGKILKYTENLNMFYLILKLLNRKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE >G (SEQ ID NO: 59)MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEELEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEEVIQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL >H (SEQ ID NO: 60)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

For A-locus through G-locus, these genes are cloned and inserted into alow-copy plasmid. A vector that does not contain Amp resistance is used.

>A-locus (SEQ ID NO: 61)TATCCGGTCGAATCGAGAATGACGACCGCTACGTCTTGGACTACGAAGCCGTGGCCCTTGCCGATGCTCTCGGTGTGGATGTTGCCGACCTGTTCCGCAAGATCGATTGCCCCAAGAACCTGCTGCGCAGGCGGGCAGGGTAGGGGAGCGGTTTCCGGCGGAGATTTTCGGAGGCGCCGGTAACGTTATGTCGGGGAATTTGCTATACATCGACGATAATTAGTTTTGTTGATTCAGGATCGAAATGCGCTCAAACAAAGAACGTTCCGCGTTTCCCTCATGCGCTACTACGCCCACACCGCCATCTTTCGGCACGCAAACAAAGCAGATGGGTTGCCTGTCAATGGGTGATCATTGCCTGAAGTTACCATCCATCAATAATATAAATCATCCTTACTCCGAATGTCCCTCAATCGCATCTATCAAGGCCGCGTGGCGGCCGTCGAAACAGGAACGGCCTTAGCGAAAGGTAATGTCGAATGGATGCCTGCCGCAGGAGGCGACGAAGTTCTCTGGCAGCACCACGAACTTTTCCAAGCTGCCATCAACTACTATCTCGTCGCCCTGCTCGCACTCGCCGACAAAAACAATCCCGTACTTGGCCCGCTGATCAGCCAGATGGATAATCCCCAAAGCCCTTACCATGTCTGGGGAAGTTTCCGCCGCCAAGGACGTCAGCGCACAGGTCTCAGTCAAGCCGTTGCACCTTATATCACGCCGGGCAATAACGCTCCCACCCTTGACGAAGTTTTCCGCTCCATTCTTGCGGGCAACCCAACCGACCGCGCAACTTTGGACGCTGCACTCATGCAATTGCTCAAGGCTTGTGACGGCGCGGGCGCTATCCAGCAGGAAGGTCGTTCCTACTGGCCCAAATTCTGCGATCCTGACTCCACTGCCAACTTCGCGGGAGATCCGGCCATGCTCCGGCGTGAACAACACCGCCTCCTCCTTCCGCAAGTTCTCCACGATCCGGCGATTACTCACGACAGTCCTGCCCTTGGCTCGTTCGACACTTATTCGATTGCTACCCCCGACACCAGAACTCCTCAACTCACCGGCCCCAAGGCACGCGCCCGTCTTGAGCAGGCGATCACCCTCTGGCGCGTCCGTCTTCCCGAATCGGCTGCTGACTTCGATCGCCTTGCCAGTTCCCTCAAAAAAATTCCGGACGACGATTCTCGCCTTAACCTTCAGGGCTACGTCGGCAGCAGTGCGAAAGGCGAAGTTCAGGCCCGTCTTTTCGCCCTTCTGCTATTCCGTCACCTGGAGCGTTCCTCCTTTACGCTTGGCCTTCTCCGTTCCGCCACCCCGCCGCCCAAGAACGCTGAAACACCTCCTCCCGCCGGCGTTCCTTTACCTGCGGCGTCCGCAGCCGATCCGGTGCGGATAGCCCGTGGCAAACGCAGTTTTGTTTTTCGCGCATTCACCAGTCTCCCCTGCTGGCATGGCGGTGATAACATCCATCCCACCTGGAAGTCATTCGACATCGCAGCGTTCAAATATGCCCTCACGGTCATCAACCAGATCGAGGAAAAGACGAAAGAACGCCAAAAAGAATGTGCGGAACTTGAAACTGATTTCGACTACATGCACGGACGGCTCGCCAAGATTCCGGTAAAATACACGACCGGCGAAGCCGAACCGCCCCCCATTCTCGCAAACGATCTCCGCATCCCCCTCCTCCGCGAACTTCTCCAGAATATCAAGGTCGACACCGCACTCACCGATGGCGAAGCCGTCTCCTATGGTCTCCAACGCCGCACCATTCGCGGTTTCCGCGAGCTGCGCCGCATCTGGCGCGGCCATGCCCCCGCTGGCACGGTCTTTTCCAGCGAGTTGAAAGAAAAACTAGCCGGCGAACTCCGCCAGTTCCAGACCGACAACTCCACCACCATCGGCAGCGTCCAACTCTTCAACGAACTCATCCAAAACCCGAAATACTGGCCCATCTGGCAGGCTCCTGACGTCGAAACCGCCCGCCAATGGGCCGATGCCGGTTTTGCCGACGATCCGCTCGCCGCCCTTGTGCAAGAAGCCGAACTCCAGGAAGACATCGACGCCCTCAAGGCTCCAGTCAAACTCACTCCGGCCGATCCTGAGTATTCAAGAAGGCAATACGATTTCAATGCCGTCAGCAAATTCGGGGCCGGCTCCCGCTCCGCCAATCGCCACGAACCCGGGCAGACGGAGCGCGGCCACAACACCTTTACCACCGAAATCGCCGCCCGTAACGCGGCGGACGGGAACCGCTGGCGGGCAACCCACGTCCGCATCCATTACTCCGCTCCCCGCCTTCTTCGTGACGGACTCCGCCGACCTGACACCGACGGCAACGAAGCCCTGGAAGCCGTCCCTTGGCTCCAGCCCATGATGGAAGCCCTCGCCCCTCTCCCGACGCTTCCGCAAGACCTCACAGGCATGCCGGTCTTCCTCATGCCCGACGTCACCCTTTCCGGTGAGCGTCGCATCCTCCTCAATCTTCCTGTCACCCTCGAACCAGCCGCTCTTGTCGAACAACTGGGCAACGCCGGTCGCTGGCAAAACCAGTTCTTCGGCTCCCGCGAAGATCCATTCGCTCTCCGATGGCCCGCCGACGGTGCTGTAAAAACCGCCAAGGGGAAAACCCACATACCTTGGCACCAGGACCGCGATCACTTCACCGTACTCGGCGTGGATCTCGGCACGCGCGATGCCGGGGCGCTCGCTCTTCTCAACGTCACTGCGCAAAAACCGGCCAAGCCGGTCCACCGCATCATTGGTGAGGCCGACGGACGCACCTGGTATGCCAGCCTTGCCGACGCTCGCATGATCCGCCTGCCCGGGGAGGATGCCCGGCTCTTTGTCCGGGGAAAACTCGTTCAGGAACCCTATGGTGAACGCGGGCGAAACGCGTCTCTTCTCGAATGGGAAGACGCCCGCAATATCATCCTTCGCCTTGGCCAAAATCCCGACGAACTCCTCGGCGCCGATCCCCGGCGCCATTCGTATCCGGAAATAAACGATAAACTTCTCGTCGCCCTTCGCCGCGCTCAGGCCCGTCTTGCCCGTCTCCAGAACCGGAGCTGGCGGTTGCGCGACCTTGCAGAATCGGACAAGGCCCTTGATGAAATCCATGCCGAGCGTGCCGGGGAGAAGCCTTCTCCGCTTCCGCCCTTGGCTCGCGACGATGCCATCAAAAGCACCGACGAAGCCCTCCTTTCCCAGCGTGACATCATCCGGCGATCCTTCGTTCAGATCGCCAACTTGATCCTTCCCCTTCGCGGACGCCGATGGGAATGGCGGCCCCATGTCGAGGTCCCGGATTGCCACATCCTTGCGCAGAGCGATCCCGGTACGGATGACACCAAGCGTCTTGTCGCCGGACAACGCGGCATCTCTCACGAGCGTATCGAGCAAATCGAAGAACTCCGTCGTCGCTGCCAATCCCTCAACCGTGCCCTGCGTCACAAACCCGGAGAGCGTCCCGTGCTCGGACGCCCCGCCAAGGGCGAGGAAATCGCCGATCCCTGTCCCGCGCTCCTCGAAAAGATCAACCGTCTCCGGGACCAGCGCGTTGACCAAACCGCGCATGCCATCCTCGCCGCCGCTCTCGGTGTTCGACTCCGCGCCCCCTCAAAAGACCGCGCCGAACGCCGCCATCGCGACATCCATGGCGAATACGAACGCTTTCGTGCGCCCGCTGATTTTGTCGTCATCGAAAACCTCTCCCGTTATCTCAGCTCGCAGGATCGTGCTCGTAGTGAAAACACCCGTCTCATGCAGTGGTGCCATCGCCAGATCGTGCAAAAACTCCGTCAGCTCTGCGAGACCTACGGCATCCCCGTCCTCGCCGTCCCGGCGGCCTACTCATCGCGTTTTTCTTCCCGGGACGGCTCGGCCGGATTCCGGGCCGTCCATCTGACACCGGACCACCGTCACCGGATGCCATGGAGCCGCATCCTCGCCCGCCTCAAGGCCCACGAGGAAGACGGAAAAAGACTCGAAAAGACGGTGCTCGACGAGGCTCGCGCCGTCCGGGGACTCTTTGACCGGCTCGACCGGTTCAACGCCGGGCATGTCCCGGGAAAACCTTGGCGCACGCTCCTCGCGCCGCTCCCCGGCGGCCCTGTGTTTGTCCCCCTCGGGGACGCCACACCCATGCAGGCCGATCTGAACGCCGCCATCAACATCGCCCTCCGGGGCATCGCGGCTCCCGACCGCCACGACATCCATCACCGGCTCCGTGCCGAAAACAAAAAACGCATCCTGAGCTTGCGTCTCGGCACTCAGCGCGAGAAAGCCCGCTGGCCTGGAGGAGCTCCGGCGGTGACACTCTCCACTCCGAACAACGGCGCCTCTCCCGAAGATTCCGATGCGTTGCCCGAACGGGTATCCAACCTGTTTGTGGACATCGCCGGTGTCGCCAACTTCGAGCGAGTCACGATCGAAGGAGTCTCGCAAAAATTCGCCACCGGGCGTGGCCTTTGGGCCTCCGTCAAGCAACGTGCATGGAACCGCGTTGCCAGACTCAACGAGACAGTAACAGATAACAACAGGAACGAAGAGGAGGACGACATTCCGATGTAACCATTGCTTCATTACATCTGAGTCTCCCCTCAATCCCTCTGCCCCATGCGTGATATAACCTCCACCTCATGTCCCGGATCGGCGCCGGCAACCTGTAGTTCCCTTCCATCCTCCAACACTCCCGCAGATCGCGATCCGCTGCCGCCGATGCCGGTGCGCCGCCTTCACAACTATCTCTACTGTCCGCGGCTTTTTTATCTCCAGTGGGTCGAGAATCTCTTTGAGGAAAATGCCGACACCATTGCCGGCAGCGCCGTGCATCGTCACGCCGACAAACCTACGCGTTACGATGATGAAAAAGCCGAGGCACTTCGCACTGGTCTCCCTGAAGGCGCGCACATACGCAGCCTTCGCCTGGAAAACGCCCAACTCGGTCTCGTTGGCGTGGTGGATATCGTGGAGGGAGGCCCCGACGGACTCGAACTCGTCGACTACAAAAAAGGTTCCGCCTTCCGCCTCGACGACGGCACGCTCGCTCCCAAGGAAAACGACACCGTGCAACTTGCCGCCTACGCTCTTCTCCTGGCTGCCGATGGTGCGCGCGTTGCGCCCATGGCGACGGTCTATTACGCTGCCGATCGCCGGCGTGTCACCTTCCCGCTCGATGACGCCCTCTACGCCCGCACCCGTTCCGCCCTCGAAGAGGCCCGCGCCGTTGCAACCTCGGGGCGCATACCTCCGCCGCTCGTCTCTGACGTCCGCTGCCTCCATTGTTCCTCCTATGCGCTTTGCCTTCCCCGCGAGTCCGCCTGGTGGTGCCGCCATCGCAGCACGCCGCGGGGAGCCGGCCACACCCCCATGTTGCCGGGCTTTGAGGATGACGCCGCCGCCATTCACCAAATCTCCGAACCTGACACCGAGCCACCACCCGATCTTGCCAGCCAGCCTCCCCGTCCCCCGCGGCTCGATGGAGAATTGTTGGTTGTCCAGACTCCGGGAGCGATGATCGGACAAAGCGGCGGTGAGTTTACCGTGTCCGTCAAGGGTGAGGTTTTGCGCAAGCTTCCGGTTCATCAACTCCGGGCCATTTACGTTTACGGAGCCGTGCAACTCACGGCGCATGCTGTGCAGACCGCCCTTGAGGAGGATATCGACGTCTCCTATTTTGCGCCCAGCGGCCGCTTTCTTGGCCTCCTCCGCGGCCTGCCCGCATCCGGCGTGGATGCGCGTCTCGGGCAATACACCCTGTTTCGCGAACCCTTTGGCCGTCTCCGTCTCGCCTGCGAGGCGATTCGGGCCAAGATCCATAACCAGCGCGTCCTCCTCATGCGTAACGGCGAGCCCGGGGAGGGCGTCTTGCGCGAACTCGCCCGTCTGCGCGACGCCACCAGTGAGGCGACTTCGCTCGACGAACTCCTCGGCATCGAGGGCATCGCCGCGCATTTCTATTTCCAGTATTTTCCCACCATGCTGAAAGAACGGGCGGCCTGGGCCTTTGATTTTTCCGGACGCAATCGCCGCCCGCCGCGCGACCCGGTCAACGCCCTGCTTTCGTTCGGTTACAGCGTGTTGTCCAAGGAACTTGCCGGCGTCTGCCACGCTGTTGGCCTAGACCCGTTTTTCGGCTTCATGCACCAGCCGCGTTACGGGCGCCCCGCACTCGCTCTCGATCTGATGGAGGAGTTTCGCCCTCTCATCGCCGACAGTGTTGCCCTGAATCTCATCAACCGTGGCGAACTCGACGAAGGGGACTTTATCCGGTCGGCCAATGGCACCGCGCTCAATGATCGGGGCCGCCGGCGTTTTTGGGAGGCATGGTTCCGGCGTCTCGACAGCGAAGTCAGCCATCCTGAATTTGGTTACAAGATGAGCTATCGACGGATGCTTGAAGTGCAGGCGCGCCAGCTATGGCGCTATGTGCGCGGTGACGCCTTCCGCTACCACGGATTCACCACCCGTTGATTCCGATGTCAGATCCCCGCCGCCGTTATCTTGTGTGTTACGACATCGCCAATCCGAAGCGATTGCGCCAAGTGGCCAAGCTGCTGGAGAGCTATGGCACGCGTCTGCAATACTCGGTTTTCGAATGTCCTTTGGACGATCTTCGTCTTGAACAGGCGAAGGCTGATTTGCGCGACACGATTAATGCCGACCAAGACCAGGTGTTATTTGTTTCGCTTGGCCCCGAAGCCAACGATGCCACGTTGATCATCGCCACGCTTGGGCTCCCTTATACCGTGCGCTCGCGAGTGACGATTATCTGACCCATAACCCACGTGTTGAAGAGGCTGAAAACAGACGGACCTCTATGAAGAACAATTGACGTTTTGGCCGAACTCAGCAGACCTTTATGCGGCTAAGGCCAATGATCATCCATCCTACCGCCATTGGGCTGGAGACGTTTTTTGAAACGGCGAGTGCTGCGGATAGCGAGTTTCTCTTGGGGAGGCGCTCGCGGCCACTTTTACAGAGGAGATGTTCGGGCGAACTGGCCGACCTAACAAGGCGTACCCGGCTCAAAATCGAGGCACGCTCGCACGGGATGATGTAATTCGTTGTTTTTCAGCATACCGTGCGAGCACGGGCCGCAGCGAATGCCGTTTCACGAATCGTCAGGCGGCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGCCGCAGCGAATGCCGTTTCACGAATCGTCAGGCGGGCAGTGGATGTTTTTCCATGAGGCGAAGAATTTCATCGCCGCAGTGAATGCCGTTTCACCATTGATGAAGAATGCGAGGTGAAAACAGAGAAATTGGGTCAACTCTATCACTCTTATTCAGCCATCGTTTCAAGAAAGGATACCTCGTATTGGATACAACACAGCTCGTTCGTTCTCTCTACCTCCCTCGACAATCTCAAGGA >B-locus (SEQ ID NO: 62)TAATAAAATTGAAATATCACTATGGATTATTGTAATATTACCATAAAGATAGGTGACGTTTTTTTGAAAATTGTAAACCTAATTTGAAGAAAACCAATTAAAAATCGCTTCGGCTTTTTTTTAAGTGCCAGGTAGCATTGATGCTAACCCATGTGTAATAAAGGTTTGTTTTCCTTCGGGGCACGAACACATTATAAGGGAAACCTAAAGATTCCCTTTCTTGTTTAATATTATAACCAGTGAAAATAAGAATAATGCACCTAAAACTAATATACAGAAAATAAGAATTAAAAGTACTAATATATACATCATATGTTATCCTCCAATGCTTTATTTTTTAATAATTGATGTTAGTATTAGTTTTATTTTAATTTCTAAACATAAGAATTTGAAAAGGATGTGTTTATTATGGCGACACGCAGTTTTATTTTAAAAATTGAACCAAATGAAGAAGTTAAAAAGGGATTATGGAAGACGCATGAGGTATTGAATCATGGAATTGCCTACTACATGAATATTCTGAAACTAATTAGACAGGAAGCTATTTATGAACATCATGAACAAGATCCTAAAAATCCGAAAAAAGTTTCAAAAGCAGAAATACAAGCCGAGTTATGGGATTTTGTTTTAAAAATGCAAAAATGTAATAGTTTTACACATGAAGTTGACAAAGATGTTGTTTTTAACATCCTGCGTGAACTATATGAAGAGTTGGTCCCTAGTTCAGTCGAGAAAAAGGGTGAAGCCAATCAATTATCGAATAAGTTTCTGTACCCGCTAGTTGATCCGAACAGTCAAAGTGGGAAAGGGACGGCATCATCCGGACGTAAACCTCGGTGGTATAATTTAAAAATAGCAGGCGACCCATCGTGGGAGGAAGAAAAGAAAAAATGGGAAGAGGATAAAAAGAAAGATCCCCTTGCTAAAATCTTAGGTAAGTTAGCAGAATATGGGCTTATTCCGCTATTTATTCCATTTACTGACAGCAACGAACCAATTGTAAAAGAAATTAAATGGATGGAAAAAAGTCGTAATCAAAGTGTCCGGCGACTTGATAAGGATATGTTTATCCAAGCATTAGAGCGTTTTCTTTCATGGGAAAGCTGGAACCTTAAAGTAAAGGAAGAGTATGAAAAAGTTGAAAAGGAACACAAAACACTAGAGGAAAGGATAAAAGAGGACATTCAAGCATTTAAATCCCTTGAACAATATGAAAAAGAACGGCAGGAGCAACTTCTTAGAGATACATTGAATACAAATGAATACCGATTAAGCAAAAGAGGATTACGTGGTTGGCGTGAAATTATCCAAAAATGGCTAAAGATGGATGAAAATGAACCATCAGAAAAATATTTAGAAGTATTTAAAGATTATCAACGGAAACATCCACGAGAAGCCGGGGACTATTCTGTCTATGAATTTTTAAGCAAGAAAGAAAATCATTTTATTTGGCGAAATCATCCTGAATATCCTTATTTGTATGCTACATTTTGTGAAATTGACAAAAAAAAGAAAGACGCTAAGCAACAGGCAACTTTTACTTTGGCTGACCCGATTAACCATCCGTTATGGGTACGATTTGAAGAAAGAAGCGGTTCGAACTTAAACAAATATCGAATTTTAACAGAGCAATTACACACTGAAAAGTTAAAAAAGAAATTAACAGTTCAACTTGATCGTTTAATTTATCCAACTGAATCCGGCGGTTGGGAGGAAAAAGGTAAAGTAGATATCGTTTTGTTGCCGTCAAGACAATTTTATAATCAAATCTTCCTTGATATAGAAGAAAAGGGGAAACATGCTTTTACTTATAAGGATGAAAGTATTAAATTCCCCCTTAAAGGTACACTTGGTGGTGCAAGAGTGCAGTTTGACCGTGACCATTTGCGGAGATATCCGCATAAAGTAGAATCAGGAAATGTTGGACGGATTTATTTTAACATGACAGTAAATATTGAACCAACTGAGAGCCCTGTTAGTAAGTCTTTGAAAATACATAGGGACGATTTCCCCAAGTTCGTTAATTTTAAACCGAAAGAGCTCACCGAATGGATAAAAGATAGTAAAGGGAAAAAATTAAAAAGTGGTATAGAATCCCTTGAAATTGGTCTACGGGTGATGAGTATCGACTTAGGTCAACGTCAAGCGGCTGCTGCATCGATTTTTGAAGTAGTTGATCAGAAACCGGATATTGAAGGGAAGTTATTTTTTCCAATCAAAGGAACTGAGCTTTATGCTGTTCACCGGGCAAGTTTTAACATTAAATTACCGGGTGAAACATTAGTAAAATCACGGGAAGTATTGCGGAAAGCTCGGGAGGACAACTTAAAATTAATGAATCAAAAGTTAAACTTTCTAAGAAATGTTCTACATTTCCAACAGTTTGAAGATATCACAGAAAGAGAGAAGCGTGTAACTAAATGGATTTCTAGACAAGAAAATAGTGATGTTCCTCTTGTATATCAAGATGAGCTAATTCAAATTCGTGAATTAATGTATAAACCCTATAAAGATTGGGTTGCCTTTTTAAAACAACTCCATAAACGGCTAGAAGTCGAGATTGGCAAAGAGGTTAAGCATTGGCGAAAATCATTAAGTGACGGGAGAAAAGGTCTTTACGGAATCTCCCTAAAAAATATTGATGAAATTGATCGAACAAGGAAATTCCTTTTAAGATGGAGCTTACGTCCAACAGAACCTGGGGAAGTAAGACGCTTGGAACCAGGACAGCGTTTTGCGATTGATCAATTAAACCACCTAAATGCATTAAAAGAAGATCGATTAAAAAAGATGGCAAATACGATTATCATGCATGCCTTAGGTTACTGTTATGATGTAAGAAAGAAAAAGTGGCAGGCAAAAAATCCAGCATGTCAAATTATTTTATTTGAAGATTTATCTAACTACAATCCTTACGAGGAAAGGTCCCGTTTTGAAAACTCAAAACTGATGAAGTGGTCACGGAGAGAAATTCCACGACAAGTCGCCTTACAAGGTGAAATTTACGGATTACAAGTTGGGGAAGTAGGTGCCCAATTCAGTTCAAGATTCCATGCGAAAACCGGGTCGCCGGGAATTCGTTGCAGTGTTGTAACGAAAGAAAAATTGCAGGATAATCGCTTTTTTAAAAATTTACAAAGAGAAGGACGACTTACTCTTGATAAAATCGCAGTTTTAAAAGAAGGAGACTTATATCCAGATAAAGGTGGAGAAAAGTTTATTTCTTTATCAAAGGATCGAAAGTTGGTAACTACGCATGCTGATATTAACGCGGCCCAAAATTTACAGAAGCGTTTTTGGACAAGAACACATGGATTTTATAAAGTTTACTGCAAAGCCTATCAGGTTGATGGACAAACTGTTTATATTCCGGAGAGCAAGGACCAAAAACAAAAAATAATTGAAGAATTTGGGGAAGGCTATTTTATTTTAAAAGATGGTGTATATGAATGGGGTAATGCGGGGAAACTAAAAATTAAAAAAGGTTCCTCTAAACAATCATCGAGTGAATTAGTAGATTCGGACATACTGAAAGATTCATTTGATTTAGCAAGTGAACTTAAGGGAGAGAAACTCATGTTATATCGAGATCCGAGTGGAAACGTATTTCCTTCCGACAAGTGGATGGCAGCAGGAGTATTTTTTGGCAAATTAGAAAGAATATTGATTTCTAAGTTAACAAATCAATACTCAATATCAACAATAGAAGATGATTCTTCAAAACAATCAATGTAAAAGTTTGCCCGTATAAGAACTTAATTAATTAGGATGGTAGGATGTTACTAAATATGTCTGTAGGCATCATTCCTACTATCCGTTTTGTCCGAATATCAGAGCATTAGGTGAGGAATGGTAAGAAAGGAAAATTTATATGAACCAACCGATTCCTATTCGAATGTTAAATGAAATACAATATTGTGAGCGACTTTTTTACTTTATGCATGTCCAAAAGCTATTTGATGAGAATGCAGATACAGTTGAAGGAAGTGCACAGCATGAGCGGGCAGAAAGAAGCAAAAGACCAAGTAAAATGGGACCAAAGGAATTATGGGGTGAGGCGCCAAGAAGTCTTAAGCTTGGTGATGAGCTGTTAAATATTACCGGTGTTCTTGATGCCATAAGTCATGAAGAGAACAGTTGGATCCCGGTTGAATCAAAACACAGTTCCGCACCGGATGGATTGAACCCTTTTAAAGTAGATGGCTTTCTACTTGACGGGTCTGCATGGCCAAACGATCAAATTCAACTTTGTGCACAAGGCTTGCTCTTGAATGCCAATGGATACCCGTGTGATTATGGGTATTTATTTTATCGTGGTAATAAGAAAAAGGTGAAAATTTATTTTACTGAAGATTTAATCGCTGCCACAAAGTACTATATTAAAAAAGCACACGAGATACTAGTATTATCTGGTGATGAATCAGCTATTCCTAAGCCTTTAATTGATTCTAATAAGTGTTTTCGCTGTTCTTTAAACTATATCTGTCTTCCGGATGAAACGAACTATCTATTAGGGGCAAGTTCAACAATTCGTAAAATTGTGCCTTCAAGGACAGATGGTGGCGTTTTATATGTATCAGAGTCTGGTACAAAATTAGGAAAATCGGGTGAGGAGTTAATCATTCAGTATAAAGATGGCCAAAAGCAGGGTGTTCCTATAAAAGATATTATTCAAGTTTCGTTAATTGGAAATGTTCAATGCTCAACGCAATTACTTCATTTTTTAATGCAATCAAATATTCCTGTAAGTTATTTATCATCCCACGGTCGTTTGATTGGTGTCAGTTCATCTTTAGTTACAAAAAATGTTTTAACAAGGCAGCAACAGTTCATTAAATTTACAAATCCTGAGTTTGGACTAAATCTAGCAAAACAAATTGTTTATGCCAAGATTCGAAATCAACGAACTTTACTTAGAAGAAATGGGGGGAGTGAGGTAAAGGAGATTTTAACAGATTTAAAATCTTTAAGTGACAGTGCACTGAACGCAATATCAATAGAACAATTACGGGGTATTGAAGGGATTTCTGCAAAACATTATTTCGCAGGATTTCCGTTTATGTTGAAAAATGAATTACGTGAATTGAATTTAATGAAAGGGCGTAATAGGAGACCGCCAAAAGATCCTGTAAATGTACTTCTTTCTCTTGGTTATACTTTATTGACACGTGATATTCATGCTGCGTGTGGTTCAGTCGGATTGGATCCGATGTTTGGTTGTTACCATCGTCCAGAAGCAGGTCGACCGGCTCTAGTATTAGATGTTATGGAAACATTTCGACCACTTATTGTAGACAGTATTGTCATCCGAGCTTTGAATACGGGTGAAATCTCATTAAAAGATTTTTATATAGGAAAAGATAGTTGTCAATTATTAAAACATGGCCGCGATTCCTTTTTTGCCATTTATGAAAGAAGAATGCATGAAACTATTACCGATCCAATTTTCGGCTATAAGATTAGCTATCGCCGTATGCTCGATTTGCACATTCGAATGCTTGCAAGGTTTATTGAAGGGGAACTGCCGGAATATAAACCATTAATGACCCGGTGAGTTTGTTTATTAGGTTAAAAGAAGGTGAAGACATGCAGCAATACGTCCTTGTTTCTTATGATATTTCGGACCAAAAAAGATGGAGAAAAGTATTTAAACTGATGAAAGGATACGGAGAACATGTTCAATATTCCGTATTCATATGCCAGTTAACTGAATTACAGAAGGCAAAATTACAAGCCTCTTTAGAAGACATTATCCATCATAAGAATGACCAAGTAATGTTTGTTCACATCGGGCCAGTGAAAGATGGTCAACTATCTAAAAAAATCTCAACAATTGGGAAAGAATTTGTTCCATTGGATTTAAAGCGGCTTATATTTTGAAAAGATATAGCAAAGAAATCTTATGAAAAAAATACAAAAATATATTGTTAAAAAATAGGGAATATTATATAATGGACTTACGAGGTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCAGGGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTTTATTTCTTTTTTCTTGGATCTGAGTACGAGCACCCACATTGGACATTTCGCATGGTGGGTGCTCGTACTATAGGTAAAACAAACCTTTTTAAGAAGAATACAAAAATAACCACAATATTTTTTAAAAGGAATTTTGATGGATTTACATAACCTCTCGCAACATGCTTCTAAAACCCAAGCCCACCATAGCCCAAAACCCCCTGCGGTCCAAGAAAAAAGAAATGATACGAGGCATTAGCACCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTCCAAGAAAAAAGAAATGATACGAGGCATTAGCACAACAATATAAACGACTACTTTACCGTGTTCAAGAAAAAAGAAATGATATGAGGCATTAGCACGATGGGATGGGAGAGAGAGGACAGTTCTACTCTTGCTGTATCCAGCTTCTTTTACTTTATCCGGTATCATTTCTTCACTTCTTTCTGCACATAAAAAAGCACCTAACTATTTGGATAAGTTAAGTGCTTTTATTTCCGTTTGAAGTTGTCTATTGCTTTTTTCTTCATATCTTCAAATTTTTTCTGTTTCTCAGAGTCAACTTTACCAACTGTAATCCCTTTTCTTTTTGGCATTGGGGTATCTTTCCACCTTAGTGTGTTCATAAGGCTTATATTTATCACTCATTGTATTCCTCCAACACAATTATAATTTTTCCGTCATCCTCAATCCAACCGTCAACTGTGACAAAAGACGAATCTCTCTTAT >C-locus (SEQ ID NO: 63)GTTTCATTTGGAAAGGGAGAGCATTGGCTTTTCTCTTTGTAAATAAAGTGCAAGCTTTGTAATAAGCTTCTAGTGGAGAAGTGATTGTTTGAATCACCCAATGCACACGCACTAAAGTTAGACGAACCTATAATTCGTATTAGTAAGTATAGTACATGAAGAAAAATGCAACAAGCATTTACTCTCTTTTAAATAAAGAATTGATAGCTGTTAATATTGATAGTATATTATACCTTATAGATGTTCGATTTTTTTTGAAATTCAAAAATCATACTTAGTAAAGAAAGGAAATAACGTCATGGACAAGCGAAAGCGTAGAAGTTACGAGTTTAGGTGGGAAGCGGGAGGCACCAGTCATGGCAATCCGTAGCATAAAACTAAAACTAAAAACCCACACAGGCCCGGAAGCGCAAAACCTCCGAAAAGGAATATGGCGGACGCATCGGTTGTTAAATGAAGGCGTCGCCTATTACATGAAAATGCTCCTGCTCTTTCGTCAGGAAAGCACTGGTGAACGGCCAAAAGAAGAACTACAGGAAGAACTGATTTGTCACATACGCGAACAGCAACAACGAAATCAGGCAGATAAAAATACGCAAGCGCTTCCGCTAGATAAGGCACTGGAAGCTTTGCGCCAACTATATGAACTGCTTGTCCCCTCCTCGGTCGGACAAAGTGGCGACGCCCAGATCATCAGCCGAAAGTTTCTCAGCCCGCTCGTCGATCCGAACAGCGAAGGCGGCAAAGGTACTTCGAAGGCAGGGGCAAAACCCACTTGGCAGAAGAAAAAAGAAGCGAACGACCCAACCTGGGAACAGGATTACGAAAAATGGAAAAAAAGACGCGAGGAAGACCCAACCGCTTCTGTGATTACTACTTTGGAGGAATACGGCATTAGACCGATCTTTCCCCTGTACACGAACACCGTAACAGATATCGCGTGGTTGCCACTTCAATCCAATCAGTTTGTGCGAACCTGGGACAGAGACATGCTTCAACAAGCGATTGAAAGACTGCTCAGTTGGGAGAGCTGGAACAAACGTGTCCAGGAAGAGTATGCCAAGCTGAAAGAAAAAATGGCTCAACTGAACGAGCAACTCGAAGGCGGTCAGGAATGGATCAGCTTGCTAGAGCAGTACGAAGAAAACCGAGAGCGAGAGCTTAGGGAAAACATGACCGCTGCCAATGACAAGTATCGGATTACCAAGCGGCAAATGAAAGGCTGGAACGAGCTGTACGAGCTATGGTCAACCTTTCCCGCCAGTGCCAGTCACGAGCAATACAAAGAGGCGCTCAAGCGTGTGCAGCAGCGACTGAGAGGGCGGTTTGGGGATGCTCATTTCTTCCAGTATCTGATGGAAGAGAAGAACCGCCTGATCTGGAAGGGGAATCCGCAGCGTATCCATTATTTTGTCGCGCGCAACGAACTGACGAAACGGCTGGAGGAAGCCAAGCAAAGCGCCACGATGACGTTGCCCAATGCCAGGAAGCATCCATTGTGGGTGCGCTTCGATGCACGGGGAGGAAATTTGCAAGACTACTACTTGACGGCTGAAGCGGACAAACCGAGAAGCAGACGTTTTGTAACGTTTAGTCAGTTGATATGGCCAAGCGAATCGGGATGGATGGAAAAGAAAGACGTCGAGGTCGAGCTAGCTTTGTCCAGGCAGTTTTACCAGCAGGTGAAGTTGCTGAAAAATGACAAAGGCAAGCAGAAAATCGAGTTCAAGGATAAAGGTTCGGGCTCGACGTTTAACGGACACTTGGGGGGAGCAAAGCTACAACTGGAGCGGGGCGATTTGGAGAAGGAAGAAAAAAACTTCGAGGACGGGGAAATCGGCAGCGTTTACCTTAACGTTGTCATTGATTTCGAACCTTTGCAAGAAGTGAAAAATGGCCGCGTGCAGGCGCCGTATGGACAAGTACTGCAACTCATTCGTCGCCCCAACGAGTTTCCCAAGGTCACTACCTATAAGTCGGAGCAACTTGTTGAATGGATAAAAGCTTCGCCACAACACTCGGCTGGGGTGGAGTCGCTGGCATCCGGTTTTCGTGTAATGAGCATAGACCTTGGGCTGCGCGCGGCTGCAGCGACTTCTATTTTTTCTGTAGAAGAGAGTAGCGATAAAAATGCGGCTGATTTTTCCTACTGGATTGAAGGAACGCCGCTGGTCGCTGTCCATCAGCGGAGCTATATGCTCAGGTTGCCTGGTGAACAGGTAGAAAAACAGGTGATGGAAAAACGGGACGAGCGGTTCCAGCTACACCAACGTGTGAAGTTTCAAATCAGAGTGCTCGCCCAAATCATGCGTATGGCAAATAAGCAGTATGGAGATCGCTGGGATGAACTCGACAGCCTGAAACAAGCGGTTGAGCAGAAAAAGTCGCCGCTCGATCAAACAGACCGGACATTTTGGGAGGGGATTGTCTGCGACTTAACAAAGGTTTTGCCTCGAAACGAAGCGGACTGGGAACAAGCGGTAGTGCAAATACACCGAAAAGCAGAGGAATACGTCGGAAAAGCCGTTCAGGCATGGCGCAAGCGCTTTGCTGCTGACGAGCGAAAAGGCATCGCAGGTCTGAGCATGTGGAACATAGAAGAATTGGAGGGCTTGCGCAAGCTGTTGATTTCCTGGAGCCGCAGGACGAGGAATCCGCAGGAGGTTAATCGCTTTGAGCGAGGCCATACCAGCCACCAGCGTCTGTTGACCCATATCCAAAACGTCAAAGAGGATCGCCTGAAGCAGTTAAGTCACGCCATTGTCATGACTGCCTTGGGGTATGTTTACGACGAGCGGAAACAAGAGTGGTGCGCCGAATACCCGGCTTGCCAGGTCATTCTGTTTGAAAATCTGAGCCAGTACCGTTCTAACCTGGATCGCTCGACCAAAGAAAACTCCACCTTGATGAAGTGGGCGCATCGCAGCATTCCGAAATACGTCCACATGCAGGCGGAGCCATACGGGATTCAGATTGGCGATGTCCGGGCGGAATATTCCTCTCGTTTTTACGCCAAGACAGGAACGCCAGGCATTCGTTGTAAAAAGGTGAGAGGCCAAGACCTGCAGGGCAGACGGTTTGAGAACTTGCAGAAGAGGTTAGTCAACGAGCAATTTTTGACGGAAGAACAAGTGAAACAGCTAAGGCCCGGCGACATTGTCCCGGATGATAGCGGAGAACTGTTCATGACCTTGACAGACGGAAGCGGAAGCAAGGAGGTCGTGTTTCTCCAGGCCGATATTAACGCGGCGCACAATCTGCAAAAACGTTTTTGGCAGCGATACAATGAACTGTTCAAGGTTAGCTGCCGCGTCATCGTCCGAGACGAGGAAGAGTATCTCGTTCCCAAGACAAAATCGGTGCAGGCAAAGCTGGGCAAAGGGCTTTTTGTGAAAAAATCGGATACAGCCTGGAAAGATGTATATGTGTGGGACAGCCAGGCAAAGCTTAAAGGTAAAACAACCTTTACAGAAGAGTCTGAGTCGCCCGAACAACTGGAAGACTTTCAGGAGATCATCGAGGAAGCAGAAGAGGCGAAAGGAACATACCGTACACTGTTCCGCGATCCTAGCGGAGTCTTTTTTCCCGAATCCGTATGGTATCCCCAAAAAGATTTTTGGGGCGAGGTGAAAAGGAAGCTGTACGGAAAATTGCGGGAACGGTTTTTGACAAAGGCTCGGTAAGGGTGTGCAAGGAGAGTGAATGGCTTGTCCTGGATACCTGTCCGCATGCTAAATGAAATTCAGTATTGTGAGCGACTGTACCATATTATGCATGTGCAGGGGCTGTTTGAGGAAAGCGCAGACACGGTCGAAGGAGCAGCACAACACAAGCGTGCAGAGACACATCTGCGCAAAAGCAAGGCAGCGCCGGAAGAGATGTGGGGGGACGCTCCGTTTAGCTTGCAGCTCGGCGACCCTGTGCTTGGCATTACGGGAAAGCTGGATGCCGTCTGTCTGGAAGAAGGTAAGCAGTGGATTCCGGTAGAAGGAAAGCATTCGGCGTCGCCAGAAGGCGGGCAGATGTTCACTGTAGGCGTGTATTCGCTGGACGGTTCTGCCTGGCCCAACGACCAAATCCAATTGTGTGCGCAAGGCTTGCTGCTTCGCGCGAATGGATATGAATCCGATTATGGCTACTTATACTACCGTGGCAATAAAAAGAAGGTTCGCATTCCTTTTTCGCAGGAACTCATAGCGGCTACTCACGCCTGCATTCAAAAAGCTCATCAGCTTCGGGAAGCCGAAATTCCCCCTCCGTTGCAGGAGTCGAAAAAGTGCTTTCGATGCTCGTTAAATTACGTATGCATGCCTGACGAGACGAATTACATGTTGGGGTTGAGCGCAAACATCAGAAAGATTGTGCCCAGTCGTCCAGATGGCGGGGTACTGTATGTTACAGAGCAGGGGGCAAAACTGGGCAGAAGCGGAGAAAGCTTGACCATCACCTGCCGGGGCGAAAAGATAGACGAAATCCCGATCAAAGACTTGATTCACGTGAGCTTGATGGGGCATGTGCAATGCTCTACGCAGCTTCTGCACACCTTGATGAACTGTGGCGTCCACGTCAGCTACTTGACTACGCATGGCACATTGACAGGAATAATGACTCCCCCTTTATCGAAAAACATTCGAACAAGAGCCAAGCAGTTTATCAAATTTCAGCACGCGGAGATCGCCCTTGGAATCGCGAGAAGGGTCGTGTATGCGAAAATTTCCAATCAGCGCACGATGCTGCGCCGCAATGGCTCACCAGATAAAGCAGTTTTAAAAGAGTTAAAAGAGCTTAGAGATCGCGCGTGGGAGGCGCCATCACTGGAAATAGTGAGAGGTATCGAGGGACGTGCAGCACAGTTGTACATGCAGTTTTTCCCTACCATGTTAAAGCACCCAGTAGTAGACGGTATGGCGATCATGAACGGTCGCAACCGTCGCCCGCCCAAAGATCCGGTCAATGCGCTGCTCTCCCTCGGCTATACGCTTCTTTCACGGGATGTTTACTCCGCATGTGCCAATGTCGGACTCGATCCACTGTTCGGCTTTTTCCATACGATGGAGCCGGGCAGACCAGCTTTGGCACTCGATCTGATGGAACCGTTCCGCGCCTTGATTGCCGATAGCGTAGCGATACGTACCTTGAATACGGAGGAACTCACCCTCGGGGACTTTTATTGGGGAAAAGACAGTTGTTATTTGAAAAAGGCAGGAAGACAAACGTATTTCGCTGCCTATGAAAGACGGATGAACGAGACGCTGACGCATCCGCAATTTGGGTATAAGCTCAGCTATCGCCGTATGCTGGAGCTGGAAGCAAGGTTTTTGGCCCGGTATCTGGATGGAGAGCTGGTGGAATATACGCCGCTCATGACAAGGTAGGAAATGACCATGCGACAATTTGTTCTGGTAAGCTATGATATTGCCGATCAAAAACGTTGGAGAAAAGTATTCAAGCTGATGAAGGGGCAAGGCGAGCACGTCCAGTACTCGGTGTTTCTGTGCCAACTCACCGAGATTCAGCAAGCCAAGCTAAAGGTAAGCCTGGCGGAGCTGGTTCACCATGGAGAAGACCAGGTCATGTTTGTAAAAATCGGCCCAGTGACGAGAGATCAACTGGACAAGCGGATATCTACTGTTGGCAGGGAGTTTCTGCCTCGCGATTTGACCAAATTTATCTATTAAGGAATGAAGAAAGCTAGTTGTAACAAAAGTGGAAAAAGAGTAAAATAAAGGTGTCAGTCGCACGCTATAGGCCATAAGTCGACTTACATATCCGTGCGTGTGCATTATGGGCCCATCCACAGGTCTATTCCCACGGATAATCACGACTTTCCACTAAGCTTTCGAATTTTATGATGCGAGCATCCTCTCAGGTCAAAAAAGCCGGGGGATGCTCGAACTCTTTGTGGGCGTAGGCTTTCCAGAGTTTTTTAGGGGAAGAGGCAGCCGATGGATAAGAGGAATGGCGATTGAATTTTGGCTTGCTCGAAAAACGGGTCTGTAAGGCTTGCGGCTGTAGGGGTTGAGTGGGAAGGAGTTCGAAAGCTTAGTGGAAAGCTTCGTGGTTAGCACCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTCGAAAGCTTAGTGGAAAGCTTCGTGGTTAGCACGCTAAAGTCCGTCTAAACTACTGAGATCTTAAATCGGCGCTCAAATAAAAAACCTCGCTAATGCGAGGTTTCAGC >D-locus (SEQ ID NO: 64)GAAGTTATGTTGATAAAATGGTTTATGAAAACGTGAGTCTGTGGTAGTATTATAAACAATGATGGAATAAAGTGTTTTTTGCGCCGCACGGCATGAATTCAGGGGTTAGCTTGGTTTTGTGTATAAATAAATGTTCTACATATTTATTTTGTTTTTTGCGCCGCAAAATGCAACTGAAAGCCGCATCTAGAGCACCCTGTAGAAGACAGGGTTTTGAGAATAGCCCGACATAGAGGGCAATAGACACGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTGAGAATAGCCCGACATAGAGGGCAATAGACTTTTGCTTCGTCACGGATGGACTTCACAATGGCAACAACGTTTTGAGAATAGCCCGACATAGTTATAGAGATGTATAAATATAACCGATAAACATTGACTAATTTGTTGAAGTCAGTGTTTATCGGTTTTTTGTGTAAATATAGGAGTTGTTAGAATGATACTTTTTGCCTAATTTTGGAACTTTATGAGGATATAAGATAGACTTGATAAAAAGGTAAAAGAAAGGTTAAAGAGCATGGCAGGAATAGTGACCTGTGATGAAGATGATGGTAGAATTAAAAGTGTTCTTAAAGAAAAACAATATTGGATAAGGAAAATAATTCAATAGATAAAAAATTTAGGGGGAAAAATGAAAATATCAAAAGTCGATCATACCAGAATGGCGGTTGCTAAAGGTAATCAACACAGGAGAGATGAGATTAGTGGGATTCTCTATAAGGATCCGACAAAGACAGGAAGTATAGATTTTGATGAACGATTCAAAAAACTGAATTGTTCGGCGAAGATACTTTATCATGTATTCAATGGAATTGCTGAGGGAAGCAATAAATACAAAAATATTGTTGATAAAGTAAATAACAATTTAGATAGGGTCTTATTTACAGGTAAGAGCTATGATCGAAAATCTATCATAGACATAGATACTGTTCTTAGAAATGTTGAGAAAATTAATGCATTTGATCGAATTTCAACAGAGGAAAGAGAACAAATAATTGACGATTTGTTAGAAATACAATTGAGGAAGGGGTTAAGGAAAGGAAAAGCTGGATTAAGAGAGGTATTACTAATTGGTGCTGGTGTAATAGTTAGAACCGATAAGAAGCAGGAAATAGCTGATTTTCTGGAGATTTTAGATGAAGATTTCAATAAGACGAATCAGGCTAAGAACATAAAATTGTCTATTGAGAATCAGGGGTTGGTGGTCTCGCCTGTATCAAGGGGAGAGGAACGGATTTTTGATGTCAGTGGCGCACAAAAGGGAAAAAGCAGCAAAAAAGCGCAGGAGAAAGAGGCACTATCTGCATTTCTGTTAGATTATGCTGATCTTGATAAGAATGTCAGGTTTGAGTATTTACGTAAAATTAGAAGACTGATAAATCTATATTTCTATGTCAAAAATGATGATGTTATGTCTTTAACTGAAATTCCGGCAGAAGTGAATCTGGAAAAAGATTTTGATATCTGGAGAGATCACGAACAAAGAAAGGAAGAGAATGGAGATTTTGTTGGATGTCCGGACATACTTTTGGCAGATCGTGATGTGAAGAAAAGTAACAGTAAGCAGGTAAAAATTGCAGAGAGGCAATTAAGGGAGTCAATACGTGAAAAAAATATAAAACGATATAGATTTAGCATAAAAACGATTGAAAAGGATGATGGAACATACTTTTTTGCAAATAAGCAGATAAGTGTATTTTGGATTCATCGCATTGAAAATGCTGTAGAACGTATATTAGGATCTATTAATGATAAAAAACTGTATAGATTACGTTTAGGATATCTAGGAGAAAAAGTATGGAAGGACATACTCAATTTTCTCAGCATAAAATACATTGCAGTAGGCAAGGCAGTATTCAATTTTGCAATGGATGATCTGCAGGAGAAGGATAGAGATATAGAACCCGGCAAGATATCAGAAAATGCAGTAAATGGATTGACTTCGTTTGATTATGAGCAAATAAAGGCAGATGAGATGCTGCAGAGAGAAGTTGCTGTTAATGTAGCATTCGCAGCAAATAATCTTGCTAGAGTAACTGTAGATATTCCGCAAAATGGAGAAAAAGAGGATATCCTTCTTTGGAATAAAAGTGACATAAAAAAATACAAAAAGAATTCAAAGAAAGGTATTCTGAAATCTATACTTCAGTTTTTTGGTGGTGCTTCAACTTGGAATATGAAAATGTTTGAGATTGCATATCATGATCAGCCAGGTGATTACGAAGAAAACTACCTATATGACATTATTCAGATCATTTACTCGCTCAGAAATAAGAGCTTTCATTTCAAGACATATGATCATGGGGATAAGAATTGGAATAGAGAACTGATAGGAAAGATGATTGAGCATGATGCTGAAAGAGTCATTTCTGTTGAGAGGGAAAAGTTTCATTCCAATAACCTGCCGATGTTTTATAAAGACGCTGATCTAAAGAAAATATTGGATCTCTTGTATAGCGATTATGCAGGACGTGCATCTCAGGTTCCGGCATTTAACACTGTCTTGGTTCGAAAGAACTTTCCGGAATTTCTTAGGAAAGATATGGGCTACAAGGTTCATTTTAACAATCCTGAAGTAGAGAATCAGTGGCACAGTGCGGTGTATTACCTATATAAAGAGATTTATTACAATCTATTTTTGAGAGATAAAGAGGTAAAGAATCTTTTTTATACTTCATTAAAAAATATAAGAAGTGAAGTTTCGGACAAAAAACAAAAGTTAGCTTCAGATGATTTTGCATCCAGGTGTGAAGAAATAGAGGATAGAAGTCTTCCGGAAATTTGTCAGATAATAATGACAGAATACAATGCGCAGAACTTTGGTAATAGAAAAGTTAAATCTCAGCGTGTTATTGAAAAAAATAAGGATATTTTCAGACATTATAAAATGCTTTTGATAAAGACTTTAGCAGGTGCTTTTTCTCTTTATTTGAAGCAGGAAAGATTTGCATTTATTGGTAAGGCAACACCTATACCATACGAAACAACCGATGTTAAGAATTTTTTGCCTGAATGGAAATCCGGAATGTATGCATCGTTTGTAGAGGAGATAAAGAATAATCTTGATCTTCAAGAATGGTATATCGTCGGACGATTCCTTAATGGGAGGATGCTCAATCAATTGGCAGGAAGCCTGCGGTCATACATACAGTATGCGGAAGATATAGAACGTCGTGCTGCAGAAAATAGGAATAAGCTTTTCTCCAAGCCTGATGAAAAGATTGAAGCATGTAAAAAAGCGGTCAGAGTGCTTGATTTGTGTATAAAAATTTCAACTAGAATATCTGCGGAATTTACTGACTATTTTGATAGTGAAGATGATTATGCAGATTATCTTGAAAAATATCTCAAGTATCAGGATGATGCCATTAAGGAATTGTCAGGATCTTCGTATGCTGCGTTGGATCATTTTTGCAACAAGGATGATCTGAAATTTGATATCTATGTAAATGCCGGACAGAAGCCTATCTTACAGAGAAATATCGTGATGGCAAAGCTTTTTGGACCAGATAACATTTTGTCTGAAGTTATGGAAAAGGTAACAGAAAGTGCCATACGAGAATACTATGACTATCTGAAGAAAGTTTCAGGATATCGGGTAAGGGGAAAATGTAGTACAGAGAAAGAACAGGAAGATCTGCTAAAGTTCCAAAGATTGAAAAACGCAGTAGAATTCCGGGATGTTACTGAATATGCTGAGGTTATTAATGAGCTTTTAGGACAGTTGATAAGTTGGTCATATCTTAGGGAGAGGGATCTATTATATTTCCAGCTGGGATTCCATTACATGTGTCTGAAAAACAAATCTTTCAAACCGGCAGAATATGTGGATATTCGTAGAAATAATGGTACGATTATACATAATGCGATACTTTACCAGATTGTTTCGATGTATATTAATGGACTGGATTTCTATAGTTGTGATAAAGAAGGGAAAACGCTCAAACCAATTGAAACAGGAAAGGGCGTAGGAAGTAAGATAGGACAATTTATAAAGTATTCCCAGTATTTATACAATGATCCGTCATATAAGCTTGAGATCTATAATGCAGGATTAGAAGTTTTTGAAAACATTGATGAACATGATAATATTACAGATCTTAGAAAGTATGTGGATCATTTTAAGTATTATGCATATGGTAATAAAATGAGCCTGCTTGATCTGTATAGTGAATTCTTCGATCGTTTCTTTACATATGATATGAAGTATCAGAAGAATGTAGTGAATGTGTTGGAGAATATCCTTTTAAGGCATTTTGTAATTTTCTATCCGAAGTTTGGATCAGGAAAAAAAGATGTTGGAATTAGGGATTGTAAAAAAGAAAGAGCTCAGATTGAAATAAGTGAGCAGAGCCTCACATCGGAAGACTTCATGTTTAAGCTTGACGACAAAGCAGGAGAAGAAGCAAAGAAGTTTCCGGCAAGGGATGAACGTTATCTCCAGACAATAGCCAAGTTGCTCTATTATCCTAACGAAATTGAGGATATGAACAGATTCATGAAGAAAGGAGAAACGATAAATAAAAAAGTTCAGTTTAATAGAAAAAAGAAGATAACCAGGAAACAAAAGAATAATTCATCAAACGAGGTATTGTCTTCAACTATGGGTTATTTATTTAAGAACATTAAATTGTAAAAAAGATTCGTTGTAGATAATTGATAGGTAAAAGCTGACCGGAGCCTTTGGCTCCGGACAGTTGTATATAAGAGGATATTAATGACTGAAAATGATTTTTGTTGGAAGTCAGTTTTTTCTGTGGAAAGCGAAATCGAATATGATGAGTATGCATATGGCAGAAGAGCTGTAGAAGGCGAGAATACATATGATTACATTACTAAGGAAGAAAGACCGGAACTTAATGACGAATATGTAGCGAGACGTTGCATTTTCGGTAAAAAAGCAGGAAAAATATCCAGGTCGGATTTTAGTAGGATAAGATCTGCGTTGGATCATGCGATGATAAATAATACACATACAGCATTTGCCAGATTTATCACTGAAAATCTGACGAGACTCAATCACAAAGAACATTTTCTGAATGTGACACGTGCATATTCTAAACCTGATTCTGAAAAATTGATACAACCGAGATACTGGCAGTCGCCTGTAGTTCCAAAGGATAAACAAATATATTATAGCAAGAATGCGATTAAAAAATGGTGTGGTTACGAAGATGATATTCCGCCTCGTTCTGTGATAGTTCAGATGTGTCTATTGTGGGGGACTGATCATGAAGAGGCAGATCATATCCTTCGCAGTTCAGGATACGCGGCGCTTAGTCCTGTTGTACTTCGAGATCTTATCTATATGTATTATCTGGATCATCAGGATTTGCAAAAAAATGAGTTGATATGGGAAGTAAAAAAGCAGTTGGATCACTTCGATTTGACAAATAGAAATTATGATACAAATCCTTTTGATGTAGGGGGCAGCGTAAATGATCATATCTGTGAACTGAGCGAGCATATAGCGAAGGCTCATTATATTTATGAGAGGGCTAAGGAAGGACCATTGCAAAATGTAATTCGGGATATTTTGGGAGATACACCTGCCCTTTATTCTGAAATGGCATTTCCTCAGCTAGCATCTATAAACAGGTGTGCTTGCAATTCGCTTTCTTCATATCAAAAAAATATTTTTGATACTGACATAGCTATATATGCAGATGAAAAGGACACAAGAGGTAAATCAGACCGTATCCTTGTTGAGGGCGCATCTTCGAAATGGTATGAATTGAAGAAACGCGATGCTAATAATGTCAAAATTTCTGAAAAGCTGAGTATACTCAATACTATTCTTAAATTTAATAGTGTTTTTTGGGAAGAATGTTACCTTGATGGAAATATAAAACAATCGAGCGGAAAGCGATCTGAGGCAGGAAAAATTCTTTATGGTCGCGACAACGGAAAAGAAAATGTCGGAGTTTCAAAATTGGAATTGGTGCGGTATATGATAGCTGCAGGTCAGGAACAAAATCTGGGAAATTACCTGGTGAGTTCAGGATTTTGGAGAAAAAATCATATGCTGTCATTTATACAAGGCAATGATATAGCGCTTGATGAGATGGATGAATTGGATCTCTTAGACTATATTCTGATATATGCATGGGGATTTAGGGAAAATATCATTAAAAAGAACAGTAATGTGAATTCTTTGGATGAAAAGACTAGAAAAGTGCAGTTTCCGTTTATAAAGTTACTCATGGCAATTGCAAGAGATATCCAGATACTTATATGTTCAGCACATGAAAAAACAGTCGATGAGTCATCTCGAAATGCAGCAAAGAAGATAGATATATTGGGAAATTATATTCCTTTTCAGATTCATCTTCAGAGAACTAAAAAAGATGGTGGAAGAGTGGTAATGGATACATTGTGTGCTGATTGGATTGCGGATTATGAATGGTACATTGATCTTGAGAAAGGAACACTTGGATGAGCAGTGATGAAAGGATATTTAAAAAATTTTTGGAAAAAGGATCGATTTCTGAGCAGAAAAAGATGCTTTTAGAAGAAAAGAAATGTTCGGATAAACTAACTGCACTGCTTGGGAATTACTGCATACCGATAGACAATATTTCAGAGTCAGACGGAAAAATATATGCGGTCTATAAGCTTCCAAAAAATGTTAAACCTTTGTCCGAAATCATTAATGATGTATCCTTTTCTGATTGTACGATGAGAGTACGTTTGCTTCTCATAAAGAGAATTCTGGAACTCGTGTGTGCTTTTCACGAAAAAAAATGGTATTGTCTCAGTATTTCACCGGGAATGCTCATGGTTGAAGATTTTGATATACCGATGGGAAATGTCGGAAAAGTATTGATATATGATTTCAGAAATCCTGTTCCGTTCGAGTCAGTAAATGAAAGACATAATTTTAACGTTTCAAATAAATACACTTCACCGGAGCTGCTCATCCATTCAAGATATGACGAGTCGAAATCTGTGAGTGAAAAATCAGATTTGTATTCTGTTGCAAAAATTGCGGAAACAATAATAGGAGATTTTAACAGTATTATTGCAAATGGAAATTTGATACTACTTGCAATGCTTAGAGTTTTTATCAGTACAGGGAAAAGTCCGGAACCTGAGTATCGGTTTGAATCGTCGGAAAATATGCTTTCAGTATTTGAAAATTTGATCAAAGAAAATTGTTTTTTTGAAAAAAACGATTATACATCTATGTTTCATCAGGCGTATGACAATTTTTTTGAATGGCAGGAATGTTTGATATCACCGGATCACTTGGATAAAAATATGTTCGAGGCAGCTTTATCAAATCTTGAGGATCAGCTGCTTAGGGTTGATATTGATAAGTATAGAGCAGAGTACTTCTATAAGCTTCTCCGAGAGTTGTCTAATAAATATAAAAATACAATTACTGATGAACAAAAGGTAAGGTTGGCAATACTTGGAATCAGAGCGAAAAATAATCTGGGAAAAAGTTTTGATGCATTGGAAATATATGAGTCAGTACGTGATTTAGAAACTATGTTGGAGGAGATGGCAGAGCTTAGTCCTGTCATTGCTTCGACATATATGGATTGCTACCGATATGCAGATGCGCAGAAAGTGGCGGAAGAAAACATTATCAGGCTTCATAATAGTAATATTCGTATGGAGAAAAAAAGAATACTGCTTGGAAGGTCATATAGTTCAAAAGGGTGCAGCATGGGGTTTCAGCATATTCTTGGTGCGGATGAGTCATTTGAACAGGCTTTATATTTCTTTAACGAAAAGGACAATTTTTGGAAAGAAATATTTGAGAGCAGAAATTTAGAGGACAGCGATAGACTTATAAAGTCTTTACGAAGCAATACGCATATTACGCTGTTTCATTACATGCAATATGCATGTGAAACAAGGAGAAAGGAATTATATGGAGCACTTTCAGACAAATATTTTATAGGTAAAGAATGGACAGAAAGACTCAAAGCATATATAAGCAACAAGGATATATGGAAAAACTATTATGAGATATATATTCTGCTAAAGGGTATTTATTGCTTCTATCCAGAAGTCATGTGTTCGTCTGCGTTTTATGATGAAATCCAAAAAATGTACGATCTTGAATTTGAAAAGGAAAAAATGTTTTACCCATTGAGTCTGATAGAACTGTATCTTGCTCTGATAGAGATAAAAGTTAATGGGAGTCTGACGGAGAATGCCGAGAAGTTGTTTAAACAGGCATTGACACATGACAATGAAGTCAAAAAAGGAAATATGAATATTCAGACCGCCATTTGGTATCGAATATATGCACTGTATAACGATGTAAAAGATGAAACTGATAAGAATAAAAGGCTTTTAAAACGGCTTATGATTCTTTGCCGACGATTTGGTTGGGCGGATATGTATAGTGCTTTGGAGAAGGATGGGAAGTTAATTGATTTTTTGAGATTTGAGGTATGTTAAATGATAACACTTGCATTAGATGAAAATGGCAAATTTGAAGATGCTTTTTCTAAAAAAAATGAAAAACCGATAATGATTGCGGGGATAATCTATGATGACAAGGGGAAAGAGTATGATGCTGAGAATGAACGCTACAGGATATCCAGTTATCTGCGAGCAGTATGTGACAGTTTGGGTGCGAAATACCCTCAGGATCTACATTCAAATAGTAATGGAAATAAGGCGACTGTTGGGAAAGTAAAATGTAAAATTGGTGAAACACTAAAGGAATTCTTGAGAGAAGGAACCTATGAAAAAAAGGAATTGCCGACAAAGAACGGTTATTTAAATAAGAGATCTGGAAAATATGTAATGTTTGCAGAACTCAGGAGTAGTCAGGGAGTTAAAAAGCGTGTTAGTGGTTGGAATGACAATGATCTGACTCAGGATGAAAAGGTCAGCAATCTGTACCTTCATATGGCAGAAAATGCCGTTGTCAGAATGCTCTTCCATAATCCTATATATGAAGATGTAACAGATGTAAATCTCTATTTTCCCACGCGAAAAGTTGTTCTGAAAGATAGAGATAGAGAATACGATAAACAAGATTTCAAAATATATGGTGATAAGGACAAGTGCGAAGCAGAAAGCGGGAGATTGGTGCATTATGATATCGTGTCATCGGATTTTTACCGTACGATAATGGAGAACGAATGTACAAGAATTAATAAAAAGCAATTAAATGTTCATTATATGAACACAAGCCCAATTTCGTACTGGGAGAAAAATGAAAAATATAATACATTTTTATATTTGGCTGACATAGTTTGTTCTATGCTGGATTATTACAAAAAGGGTTCGAGTCCGGCAGAGTGGATGGATTCTTTTGCCGAATGGGGAAACAAATATTTTGGTGATGATCAGATAATCTTATTTGGGTATGATGATATAGATGACAAATACATGGAGGCTGTAGATGCAGTAGGACAGGGAGAGTATTTTCATGCGCTGGATATTATATATGATGCGGAATGTAGTGGAAGTGAATTTGAGAAGCACTACAAAGATTATTGGTTTCCAAAGCTTATAAAAAAGATACGAATAACAGCAACTGTGGATAATTTATGCAGATCGATCTCAGATCTGGAGAGTTTTACATATCGAAGTAATCTTGATCAGCAGAAACTTTTGTGGATTTTTGAGGAAATCAAAGCTATCGTCGATAAGGGAGATTTTGGAAAGAAATATCATACAGATCAGGTTATGTTTGATATGTGTAATGCCGGTATTGCTGTGTACAATCATATCGGAGATTTTGGGACTGCAAAGGAATACTATGATGAGTGCATGAAACACACTGGGGATGTGGATCTGGTAAAGATACTTCGTGCATCAAATAAAATGGTGGTCTTTCTTGACGATGCTTTTAGGTATGGTGACGCGACAGAACGTGCCAGGAAGAATGTTGAATACCAAAAAGCTTTGCACGATATAAAGAGTGAGATTTGTCCGGAAAAGAAAGATGAAGACTTGAACTATGCCATATCGCTCAGTCAATTTGGACAGGCGCTTGCGTGTGAAAAAAATTCTGATGCAGAGAGTGTTTTCCTAGAGTCGTTGCGGCATATGAGGAAAGGGACTGCCAATTATCAGATTACTCTTTCATATTTACTCCATTTTTATCTGGATATGGGAATGACAGATTCTTATCGAGAAAAAACAAAGGACTATTTTGGAAGTGAAAAACCAAAGGAACAGCTGAAAGAATTGCTGAAGTTATCGGGAAAGGATGATAGTATAGTTACTTTCAAATTTGCAATGTATGTCTATTTACGTGCACTTTGGGTATTACAGGAACCGCTTACTGATTTTATCAGAACAAGATTAGAGGACATACGTGAGACTCTTGTAAAGAAGAAAATGAGTGAACATATGGTTGGACATCCGTGGGAGTTGATTTATAAATATCTGGCATTTCTTTTTTATCGTGATGGAAATTGTGAAGCTGCTGAAAAATATATTCATAAAAGTGAAGAGTGCTTGGAAACACAAGGACTGACTATAGATGCGATTATTCATAATGGTAAGTATGAATATGCAGAATTGTCAGGTGACGAGGAGATGATGGCAAGAGAGAAAGCGTACTTTGATGAAAAAGGGATAGATAGAAAAAATGTTTGTACTTTTATGTATCATTGATGTTTAATAAGATTTGACCGAGGAGTGACAGGTAATCGCCGGTATATCTGGTATTACCTGTCATTTTTTGATGAAATAAGCTACTTTTTGCCTAAAAAACGAAACTGTTGGTGTTTTATGATGATTGTGTCAACAAAAGAGAGCAAAAGAAGAGGAGAAAAGTAATGTCAATGATTTCATGTCCGAATTGTGGTGGAGAGATATCTGAAAGGTCAAAGAAATGTGTTCATTGTGGATATGTGTTAGTCGAAGAAGCTAAAGTAGTGTGCACAGAATGTGGAACTGAGGTAGAGAGTGGCGCTGCTGTATGTCCGAAGTGCGGCTGTCCTGTAAATGATAGTGAGACGCCTCAGAAAGTTGAAGTGACTAGGGTAAATGTATCTTCCGTAATCAGCAAAAAAGTCGTTGTAAGCATACTGATCGCAGTGATTACAATTGCAGGTTTTTTCTATGGAGTGAAGTATTCGCAGGAAAAGAAAGCAATTGAAGAGTCAGTAAAGCAGAAGGAAGACTATCAAAGTACGCTAGAGCTTGCTTCGCTAATGATGCTTCAAGGAGCTTCGGATGCAGAAACTTGTGGGAATTTGGTTAGGAAAGTGTGGAGCAACTGCATTTATAAGGAGAGGGATGAAGAAACCGACAAGTATACGTGTGATAGCAGGGGTGCAGGATGGTTTTATGATGATTTTAATGATGCATTAATGGCTCTTTACAGTGACAGCAGTTTTGGCAAGAAGATAAATGAAATCAAAAACGGTCAGGAAACCGTTGCGGCGATGATGAAAGATCTGAAAAATCCGCCGGATGAGATGGCAGATGCCTATGAGGATATTCAAAATTTTTATGTGTCCTATCTAACGCTGACAGAAATGGTTGTGAATCCAACTGGAAGTTTGAGTTCTTTTTCATCTGATTTTTCCGATGCGGATACGGAGGTGTCCAATGCCTATAGCCGGATGAAGTTGTATTTAGATTAAACTATTGAGGAAAAAATGGAGGTGCTTTAATGCGGGGGAGAAACTGTGGAGGGTCATCAGGCGACGGACTGCTGGTACTTCTCGTACTGCTTGTCCTTTTTTATAAAATCATGCCATTCATAGGTTTATGGATTTTAATTTTTGGTGATGCTGAACGTAAAGATCTGGGTATGGGTATGATTATTGTCGGGATAGTTCTATATGTATTATTAGAGGTTTTTTAATGTGAGTTTCTGTGGTAAACTATAAAAGTACAAGCTTTTGCGCCGCACCGCATAAATAGCGGATTTATGACCATTATTTGGTGAAAAAAATGGTGTACACCTGTGTTTTTTTGTTTTGCGCCGCAAAATGCGCCACGGAACCGCATGCAGAGCACCCTGCAAGAGACAGGGTTATGAAAACAGCCCGACATAGAGGGCAATAGACACGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTATGAAAACAGCCCGACATAGAGGGCAATAGACATAAAGACCAAAAACAGGTCATCTGCATACTGTGTTATGAAAACAGCCCGATATAGAGGGTGTGAGAGATATAGTTCTCGTCACAGTGCAGAAAATGACCTATTATGTGCCGAAAAACAAAATGAAAAAAGAATGGAAAGGCGTATTTAATGAAATGCTGATCTGTTGATTTGAATTAACAAAAAAAGGTCGCCCCACGGATGACAAAAACATCCGGGGGCGACCCTTTT >E-locus (SEQ ID NO: 65)TACTGTGTGCATAAGTCTTCCTTAGATCCATAGGTACAGCAGTTTTATTTATTAGCCTTAGAAAATGGAAAATAGAGCTTATAAATGATATGATATTTATGAATAAAATGATTGCATTCTCGTGCAAACTTTAAATATATTGATTATATCCTTTACATTGGTTGTTTTAATTACTATTATTAAGTAGGAATACGATATACCTCTAAATGAAAGAGGACTAAAACCCGCCAAAAGTATCAGAAAATGTTATTGCAGTAAGAGACTACCTCTATATGAAAGAGGACTAAAACTTTTAACAGTGGCCTTATTAAATGACTTCTGTAAGAGACTACCTCTATATGAAAGAGGACTAAAACGTCTAATGTGGATAAGTATAAAAACGCTTATCCATCATTTAGGTGTTTTATTTTTTTGTGATTATATGTACAATAGAAGAGAGAAAAAAATCATTGAGGTGAAAACTATGAGAATTACTAAAGTAGAGGTTGATAGAAAAAAAGTACTAATTTCTAGGGATAAAAACGGGGGCAAGTTAGTTTATGAAAATGAAATGCAAGATAATACAGAACAAATCATGCATCACAAAAAAAGTTCTTTTTACAAAAGTGTGGTAAACAAAACTATTTGTCGTCCTGAACAAAAACAAATGAAAAAATTAGTTCATGGATTATTACAAGAAAATAGTCAAGAAAAAATAAAAGTTTCAGATGTCACTAAACTTAATATCTCAAATTTCTTAAATCATCGTTTCAAAAAAAGTTTATATTATTTTCCTGAAAATAGTCCTGACAAAAGCGAAGAATACAGAATAGAAATAAATCTCTCCCAATTGTTAGAAGATAGCTTAAAAAAACAGCAAGGGACATTTATATGTTGGGAATCTTTTAGCAAAGACATGGAATTATACATTAATTGGGCGGAAAATTATATTTCATCAAAAACGAAGCTAATAAAAAAATCCATTCGAAACAATAGAATTCAATCTACTGAATCAAGAAGTGGACAACTAATGGATAGATATATGAAAGACATTTTAAATAAAAACAAACCTTTCGATATCCAATCAGTTAGCGAAAAGTACCAACTTGAAAAATTGACTAGTGCTTTAAAAGCTACTTTTAAAGAAGCGAAGAAAAACGACAAAGAGATTAACTATAAGCTTAAGTCCACTCTCCAAAACCATGAAAGACAAATAATAGAAGAATTGAAGGAAAATTCCGAACTGAACCAATTTAATATAGAAATAAGAAAACATCTTGAAACTTATTTTCCTATTAAGAAAACAAACAGAAAAGTTGGAGATATAAGGAATTTAGAAATAGGAGAAATCCAAAAAATAGTAAATCATCGGTTGAAAAATAAAATAGTTCAACGCATTCTCCAAGAAGGGAAATTAGCTTCTTATGAGATTGAATCAACAGTTAACTCTAATTCCTTACAAAAAATTAAAATTGAAGAAGCATTTGCCTTAAAGTTTATCAATGCTTGTTTATTTGCTTCTAACAATTTAAGGAATATGGTATATCCTGTTTGCAAAAAGGATATATTAATGATAGGTGAATTTAAAAATAGTTTTAAAGAAATAAAACACAAAAAATTCATTCGTCAATGGTCGCAATTCTTCTCTCAAGAAATAACTGTTGATGACATTGAATTAGCTTCATGGGGGCTGAGAGGAGCCATTGCACCAATAAGAAATGAAATAATTCATTTAAAGAAGCATAGCTGGAAAAAATTTTTTAATAACCCTACTTTCAAAGTGAAAAAAAGTAAAATAATAAATGGGAAAACGAAAGATGTTACATCTGAATTCCTTTATAAAGAAACTTTATTTAAGGATTATTTCTATAGTGAGTTAGATTCTGTTCCAGAATTGATTATTAATAAAATGGAAAGTAGCAAAATTTTAGATTATTATTCCAGTGACCAGCTTAACCAAGTTTTTACAATTCCGAATTTCGAATTATCTTTACTGACTTCGGCCGTTCCCTTTGCACCTAGCTTTAAACGAGTTTATTTGAAAGGCTTTGATTATCAGAATCAAGATGAAGCACAACCGGATTATAATCTTAAATTAAATATCTATAACGAAAAAGCCTTTAATTCGGAGGCATTTCAGGCGCAATATTCATTATTTAAAATGGTTTATTATCAAGTCTTTTTACCGCAATTCACTACAAATAACGATTTATTTAAGTCAAGTGTGGATTTTATTTTAACATTAAACAAAGAACGGAAAGGTTACGCCAAAGCATTTCAAGATATTCGAAAGATGAATAAAGATGAAAAGCCCTCAGAATATATGAGTTACATTCAGAGTCAATTAATGCTCTATCAAAAAAAGCAAGAAGAAAAAGAGAAAATTAATCATTTTGAAAAATTTATAAATCAAGTGTTTATTAAAGGTTTCAATTCTTTTATAGAAAAGAATAGATTAACCTATATTTGCCATCCAACCAAAAACACAGTGCCAGAAAATGATAATATAGAAATACCTTTCCACACGGATATGGATGATTCCAATATTGCATTTTGGCTTATGTGTAAATTATTAGATGCTAAACAACTTAGCGAATTACGTAATGAAATGATAAAATTCAGTTGTTCCTTACAATCAACTGAAGAAATAAGCACATTTACCAAGGCGCGAGAAGTGATTGGTTTAGCTCTTTTAAATGGCGAAAAAGGATGTAATGATTGGAAAGAACTTTTTGATGATAAAGAAGCTTGGAAAAAGAACATGTCCTTATATGTTTCCGAGGAATTGCTTCAATCATTGCCGTACACACAAGAAGATGGTCAAACACCTGTAATTAATCGAAGTATCGATTTAGTAAAAAAATACGGTACAGAAACAATACTAGAGAAATTATTTTCCTCCTCAGATGATTATAAAGTTTCAGCTAAAGATATCGCAAAATTACATGAATATGATGTAACGGAGAAAATAGCACAGCAAGAGAGTCTACATAAGCAATGGATAGAAAAGCCCGGTTTAGCCCGTGACTCAGCATGGACAAAAAAATACCAAAATGTGATTAATGATATTAGTAATTACCAATGGGCTAAGACAAAGGTCGAATTAACACAAGTAAGGCATCTTCATCAATTAACTATTGATTTGCTTTCAAGGTTAGCAGGATATATGTCTATCGCTGACCGTGATTTCCAGTTTTCTAGTAATTATATTTTAGAAAGAGAGAACTCTGAGTATAGAGTTACAAGTTGGATATTATTAAGTGAAAATAAAAATAAAAATAAATATAACGACTACGAATTGTATAATCTAAAAAATGCCTCTATAAAAGTATCATCAAAAAATGATCCCCAGTTAAAAGTTGATCTTAAGCAATTACGATTAACCTTAGAGTACTTAGAACTTTTTGATAACCGATTGAAAGAAAAACGAAATAACATTTCACATTTTAATTACCTTAACGGACAGTTAGGGAACTCTATTTTAGAATTATTTGACGATGCTCGAGATGTACTTTCCTATGATCGTAAACTAAAGAATGCGGTGTCTAAATCTTTGAAAGAAATTTTAAGCTCTCATGGAATGGAAGTGACATTTAAACCACTATATCAAACCAATCATCATTTAAAAATTGATAAACTCCAACCTAAAAAAATACACCACTTAGGTGAAAAAAGTACTGTTTCTTCAAATCAAGTTTCTAATGAATACTGTCAACTAGTAAGAACGCTATTAACGATGAAGTAATTCTTTTAAAGCACATTAATTACCTCTAAATGAAAAGAGGACTAAAACTGAAAGAGGACTAAAACACCAGATGTGGATAACTATATTAGTGGCTATTAAAAATTCGTCGATATTAGAGAGGAAACTTTAGATGAAGATGAAATGGAAATTAAAAGAAAATGACGTTCGCAAAGGGGTGGTGGTCATTGAGTAAAATTGACATCGGAGAAGTAACCCACTTTTTACAAGGTCTAAAGAAAAGTAACGAAAACGCCCGAAAAATGATAGAAGACATTCAATCGGCTGTCAAAGCCTACGCTGATGATACAACTTTAAAAGGAAAAGCAGTGGATTCTTCACAAAGATACTTTGATGAAACGTATACTGTTATTTGTAAAAGTATCATAGAAGCATTAGATGAAAGCGAAGAGAGATTACAACAATATATTCATGATTTTGGAGATCAAGTGGATTCTTCACCTAACGCACGAATTGATGCGGAATTACTACAAGAAGCAATGAGTAGGTTAGCTGACATAAAGCGGAAGCAAGAAGCACTTATGCAATCCTTATCTTCTTCTACAGCAACGCTTTACGAAGGCAAGCAACAAGCGTTACACACTCAATTCACGGATGCGCTGGAGCAAGAAAAAATATTGGAACGCTATATTACTTTTGAACAAACTCACGGGAATTTTTTTGACTCATTTGGAGAACTTGTCTATCGAACGGGACAAGCAGTGCGTGAATTAGCTAATAACGTCACATTCGAGAGCCAAACAGGAAGCTATCATTTTGATAAAATAGATGCTTCTAGATTCCAAACTTTGCAAGAAATGTTGCCAAAGGCAAAGAAAAAAGCATTTAATTTTAATGACTACCAAATAACATGGAATGGCACCACGCACCTTTTATGGAAAAATGGTAAAGTGGATGCAGAAGCAACCAAAGCTTATAACGAGGCGAAACTGAATGGAAAGCTACCAAAGGAAGGTAATGTAGCAACACAAGATGCAGAACTATTAAAAGGCATTTTGGCTTCACTGAAAAACAAGAAAGATCCTATCACTGGAGCAGATATAAGCAGTGTGCATGTATTATCTATCCTTAGCGGGCTCGCATTCTCCTATACAGCTGGGAATTATAAGGGAAGAAAACTTACTGTTCCAAAAAGTTTCTTAGACAAATTAAAGAAAAACCGAAAATCTAAAGTACCTAAACTATCTAGTTTATCAGAAAAACAACAACTAAAACTCGCAAATAAATACAAGAAAAAATCACCTATTCCAATTCCAGATGATGCTAAAATCAAAGCTCAGACGAAAAAGGCTGGTTATGAACAAATATCTTATAAATGGAAAGAGAATGGGATAACCTTTGAAGTTAGATGGCATACTAGGACACCAGGTGCACCAAAGGAACAAGGAAATACGTTTGTTATAGAAAGAAAAATTCAGGGTACAGCAGAAGGGAAAACAAAAGTTCAACAAATATTGGTTGGAGATAATAAGTGGGTGAGTAAAAGTGAGTGGCAAAAGGCTATAACTGATAAGAAAAATGGTGTAAGTACCTCGGAGCAAAATAAAATGTTGTCTGATGGACATTGGAAAGAATAGAAAGGAGCAAAATGATGGAAGATTATTATAAAGGTTTTGAGGGATATCCAGAGATAGATTTTTATACGTATATAGATGATATGAAATTGGGTATAGCAATGTGGGAAGGATACTTTGACAACATTATGAAAGAAATTAATCCAAGTAACGGAAGATGGACTTCATTAGCGTATTATTATCATTTAGATGAGGGGTGGTATGATGAAAGTCCTTGGGAAATACCAAGTAATACAGAAGCATTAGAATTATTGGAAACAATCCATATATCTAATCTAGATACTATCACACAAGAGATATTACTTAAATTAATAAATTTATTAAAGAAGAATATAAATAGACAAGTTTATATTGAATACTCATAAAAAAGATGATTATGATATATTATAGAACAAACGAACAAGCCCCAAATACGAGGTTTGTTCGTTTGTTTTCAATATAATTATTTGCCACCAAGTGAGATATTACGGTTTTAAATAGCTTATTTGACGATACCAAACCCTGATAAGAGAAAGAAGAAAGAGAAAGCTGGTGTAGTTGTTTTAAGTGAACTAGATAAAAAATTAATAGCAAAACTTGAAAAAGATGGTGTGAAAATATCAAAAGAAGATGTTATAGGAATAAAATAATTGCCAGATGATGAGAAATCGTTTGGCTGGAAAAAGGAAATCCATCCGCTGGATTTGAGCATATTCTTATTGAACATGGTGAACAATTTGCTAAATAGGGAATTTCAAAAGCTGAGTTACCTGATTTTTTGATGACTGCTTTAGAAAAGGAAA >F-locus (SEQ ID NO: 66)ATTCTTTAAAAATATCTAATAATTTATTTACTATATACTCTAATACATCTTTTAACCTATCTAAAACATCATCACCTACAACATCCCAAAAATCATCTAAAAAGTTAAAAAAATCCATCTTTATCAACTCCTATATCTATTTTTTATTGTGTAATTCCTGAGTTACAAAACCATTATAACACGTATTACACACGTAGTCAATACTTCAAAAAAATTTTTTGTATATTTTTTTGAATAAGTAAATAAAAAGAGCTGTGTAGCTCTTTATTAAAATCAATATTTTTATTTTGTTAACAAACTTAGACAACATTAAATTTAGAAACCTATATATATTTCAGTACTTTTCATTTTTAGGTAGTCTAAATCAGAAATGGTTTTGTCTAAATGATGTATGTAAGTTTTAGTCCCCTTCGTTTTTAGGGTAGTCTAAATCAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTAGTCCCCTTCGTTTTTAGGGTAGTCTAAATCCCATCCAAATTATGGGATAATATGTTACTTTTTATTTTAATATTTGATTATTTATTGTTTTTTTACTGATTTAGATTACCCCTTTAATTTATTTTACCATATTTTTCTCATAATGCAAACTAATATTCCAAAATTTTTGTTTCTTTTCTTATGATCTTTTCTCCGATAGTTATTTCTCCAGATAAGATTTTCATTTTTTTGAATTGATCTTCTGTTAGAATTAATGTTCTTACTGATGAATTTTCTGGAACTATCATTGACAACTGATTTTCATAGGAAATTATTTTTTCTTTTGTGCTAGAACTTACAATGTATACTGATTTTTGTACCTGATAATATCCTTTTCTTATAATTTCTTTTCTAAATTTTGCATATTCTTTTTTTTCTTTTCCTGTTTGCATTGGAAAATCATACATTAGAATCCCTACATAATTAGTACTCATAATCCTCTATCCTTAACTCAGGAATTTCTACTTCTGACATTTCTCCTGTAAAATAATTTCTAATATTATCTAAAAAATAATCAATCACTTGAGCCAATTCATATTTTTTATTTTTCCAATAAACTTTTTGTGTTAATACCAATAACAATTTTTGTCTTAATGATTTATTCAAACTTACTTCTTCCTGTTGATTAAAATATACGATATAATCTACCATTGGACGAAATATTTCAATAATATCATCTGCAAAATTATAATTATTAAATTGTGAACTGTGATGTATTCCCAAACTTGGATGAAATCCTTTAGCCACAATTTTTGAAGAGATTAAGCTTCTCAAAACCATATACCCATAATTTAATGCCGAATTTGTCCCGTCTTCACCAAATCTCTTAAATTTTTTCCCAAAAAGTTCACCAAAATACATTCTTGCAGCAATTGCTTCCTGATGTTCCGCTTCTTTTCCTTTTAATCTAATATTATTTTCATATGCTTCCAACTTATATGATACTTCCTGAGATTTTTTCAAAAACTGCAATAAATTTCTTTGATTTTCTATTTTTCTCATTACAATTTTTCTCCAGATTTCTTCTTTTTTATCGTCAATCCAGCTCACTTGCTCATTAATTCTTGTTGTTACTTGAAAATGATTATACAGTCCTAATGAATGTAAAACTGGCTGATGTTTTTCATTACAAATTATCAGTGGAATATTATGTTCTGATAATCTTAACTGTAATATTCCGCTAATTTTACATCTGCAATTTTCAACTACAATTGCCATGATATCATTTAAAGATACTTTATCAGCCTTATTTTCATCATCTTCATTTATCATCACAAGCTGGTTATTTAAAACTGATAATTCATTGACTCTTGTTACATGGATAATATTAGACATTTTTATTACTCCTTTACTCTAAAGCTTTATATTCAAACATAACTTTCACAAGTTCACACAATTCTTCTGAATTTCTATCAGTCATTAATTTTTTCTTTTTTAAATTTTTCAAATGTACAATTTTTTCCGATTCTAAAGTCTGAATTTCTATTTTCTTATCTGCTCCTATTTTAAATGTTGCTACAAAACCATATTCCTTTAATATATCCACTATTGATTTCATAATTGCATTTTTAAGTTTTCTATCATAAGAAAGTAATTTTCTTAAATTTTCCAGCACTTCTAAAAGTGAAATTTCAGCATGCGGAATATAGTTAAAATGTGCAATATAGTTTCGTATATACAAATCTTTTTTCTCTTGTTTTAATTTTTTTACTTTTTTATCAGAATAGATGCTTCTTTTTTCTACATTATCTTTGTATAATTCTTTATAAAAATTTATATATTTTTCAACAATTTGCCCACTTTTATATTTTACATTTTTACTGTTATCAAAATTAAATATTTCTTCAATATAATGATTTTCAGGAAATTCACCTTTCAATCTAAATCTTAAGTCCCTTTCCCAGATCGAAGTATATCCCACAAGTCTGTGGAGTATTTTTAATAACAAGCCTTGCAACAAGTTTAATTCATTAAATTCCACTTTATTTTTCAAATGAGTATATTTTTGTATATTTCCAATTGCTTTTTCATATTCTTTATAATCTTCATCATTAAATTTTTCATCTTTTTTAGGTCTTGCATATTTTCTATGTAAATTTTGCTGCATTGTATAATTTTTTTCTATTTCATTTTTTTTATTGCTGTATTCTTTCAATTCTTTTAAACTTATTTTATACTTCGCTTTATCAGCTATTTTTTCAAGTAAATTTAACATCCCATATTTTTTTATATTATAAAAAGCTCTATGCTTTATAATATTTTCTCCATCAAAATATATTTTATTTGTGTCAAATTTCTTCAATTCTTTCCTATCTTTTATTTTATTTTCATTAAAATCTAAAAATTTTCCAATTTCATTCGCTTCTAATTCAAAATCTTCTGTTACTCTATTATTATCTAAATTTAAAAGATTTATAAGTTCAAGTTCATCTGAAAAAGTTTCTTCTTTATTTGCACTCTGATATTTTTCAAGACTTCCCTTCAAATTAGTCAATTCTTTATGATTAAGCAATTTTAAAATTAAATAAAACATATTCAAATTTTCAGTGTATTTTAATATCTTTCCTAATTTTATCTCTCTTACAAATTCATTTATTTCATGTGGAATTTCTTTATTCCTATTATGTTTTTCATAATTTTTTAAAATTTTATCATATTTTTCTTTATTATCTTTTTTTATTTTTATTTTAGAAAATATATCATTATTATCATTGTTATTATTACTTTCTATATATTTTAAATTATTTTTATTCAAATAATCTATAAAACCTTTTAAAAATATTTGTTGTATAAAATCAATGTATGTATTTTTTTCTTCTTTATCTTGATTATTAATCATCTCCCTACTTTGTATAATAGCAAGATATTCTACTGGTACAGTTTTTTCTATATTTTCAAATTTTTGATATTTATAATGTCCTGTTTTTTGATTTCTTTGTTTATTTATTTTTATTACTTCATTAGTTATTTTAAAAAAAACTTTACTATTTTTAACAAATTTATTAAGAAATTCACCATAATAAATATTTTTCAAAAGATATATTTGAGCATCTTTTTCTTCTTTATCCTTAGGAACACTCCAAAAAAATTTTAAAGTATTTCTTAAATCTTCTATTTTATTATATAATTTCGTAAAAGAAGGAACAAAAGGAATATTCTTATTTACAAAATTAAATTTTGTATTTTTTAAATATTTAATTATCACATCCTTTTCATAATAATTAAATACATTTGCACTATTTAACTGCTTAAATATCTTCAATTTCAATTTTTTCTCATTTATTTCATTTTGAAACATTTTTTTTGAAATTTCAGAAGGAGCTATATTTTTAAATGCAAATATATCTTTCCCTTCTAATTCCAAATTAAAATGCACAATCCCATGTCTAATACTGCTAATAGCTTCATCAATATTTGCAAAAAAATCTTCTATCTCATTTTTATTATCCATATTAAAATCATAACTATAGAACATTTTTAAATTTTCTTTTACTTCATTTTGCTTGTTTTCATTATATATTTTATCAACTTCTCCAGAAACATATTTTTCTTCGCCCTTATTATTTTTTACAGTTTTTCCTCTCATTCTACCTGTAATATCATTCTCATTTTCAGTTTCAAGAATATTTCTCAATGAAAAATATGCAACCGAAGAAACTCCAATTATATTTCGTAAAAATGCTTCATTTTGTCTATTCCTAGCAATAAAATCACTTGTTGCAATCTCTCCAACTTGTAAATAATAATTGTATTTCCCACAATTTCTTACATAAGTATCCAATTTATTTAGTAATTTGTTTTCAATTAATTTTTTTAAATTTTGATATTCAAATATTCTCTTAATTTTATCGTTACTTATGTTACTCAGTCTTTTATACACATAATTTTTCAAAAGCTGACTCATTTCAATTTCCACAAAATGACAAAAAGCATATTTTATATTTTTATCATTAAGTTCTTCTTTATCCAAATAATATTTATAAAACACTTGTGATTTTTTTAATTCACTCATATCCGGAATTTTTTCAATTAATTCTTTTATATTATTTACATTTTGTATTTCTTCGTAAATAATTTTAGCAAAATTTTCTTTATCATTTTTTCTTCCAATTATTTTGTGATAGTATTCTCTTATTTTATATTTTTCATGTTTTTTTGAATTTTCTATTAAAAAAAATAACTTCTCAATATCTTCTTTTTTATACAATTTATCAAATGCTTCCTGTACATTATTTATATAATCATTACGCTTTGCTGATTCTCTATAATAATCATAAATAATATTTCTTTTGCTCTTCCCTCCAACTTTTTCAACATTATTTTCATTAATTTTCTGATAATTAGCCTTATTTTCTTCAAATGAATATTTTAAAGAATTTATCTTATTCAATTTTGCCTCAACATCTTTTCTAAATATTTCTAATTCTTCAGAGTTCACATCTTCATTTAACAATATTTTCTTTAAAACTGAAAAACTATTTTTATTTTTTAAATCATATTCTGAAATATCTTCTTCAGAATAATTTTTATCCTGTACTGCATTTTTCTCTTTCCTATTCTTTAAATACAGAACACTATCTTTTAGATGCAATACTTTATTTGAAAAAAACTTTTTTAAATTTTCTCTTCTTATTCTATTTTCTTCTTCACTTGCATTATCAGGATTTTTTATATATATATCCAGTCTTATACTTAAAAGCTCTGACAATCTCTCACTAGTCCTATTTTCTTCGCTCGTACTTTTTACTAATTTTCCCTCTTCAATATATTTTTTATGCGAAATTCCATCAACTTTTGTAACTTTCATATATAAAAACCTCCTAATATCTATATTTTTTACTCAATACCTAATTCTTTTTTCAATGCTTTTTGTAAAATTTGTGAAAAATTCAGATTTTTTTCCTGTGCCAATATATCTAACCAAACAGGAATTGTTAAAGTTTTCTTTTTAAGTGCATTTGTAACTTTTGCCACTTCATACACTGGATCAACAGATAAAATATACAAATACTGATTTTCTTTCAGTTTCACATCCTCCACTTTTGAAGGCTCAGGAAATTTTTTTCTTACATCCAAAAAATCAGCCAAATGCAGACCCAATGTCTCTCTCAAATTGGAAACAGCCTCCTCCATGCTATCTCCAAATGTAGCATAATAATTTATCTCTCCATCTTCAAACTTATCAAAATCAACAATACAACCATAATAAGTCCCATCTTCCTTAGTTACCACTGCTGGATAAAATACATCCATTTTAATTATCTCCAATCTATACCACGTGTTAAATACGTGTTTAAAAATATTTATAAAATTTTTTAGCATCTCTGCTAAAATAAAACAATTATTTCAAATTTTTCTATTCCTTAATCACTCATTGTTAGTGATTCTTTTTTTACTTGGACAATTTTTCATTTAATTTCTTCAATTTTTTTAAAATCACATTTTTTTAATATTCCTTATTTAATTGCAAATTTTCATTACTTTTGGGGTGCTCTAAATCCCATCCAAATTATGGGATAATAATTTTTAGTGAAAGCAAGAAGGGACTAGAATTTAATCCCAACTTGTTTTTCAATACTTCTTAATGTTCCTACAGGTATATCTTTTGAATATGGTACTGTGACCACACCTTCCACACCTGGGATCATCCATTGATAATGACTACCTCTTATACGCACAACTTTTCCGCCTAATTTTCTAAATCTTTTTTCGAT >G-locus (SEQ ID NO: 67)CTTTCTATCTTTTTCAAATAAAATTAGGCTCTAGTTAGCCTAATCGCATAATTATTTATTATAGTATAATTCTTATTTTTTTTCAACCTAAAAATTTAAAACATCTCCAAAAATTTTCGTTTCAGAACAACCAAGCAACCATATTCAAAAAACAATAAAAAATGAGCAAGAATTGAAATTTTATTCTCACTCAGAAGTTATTTTTATTAAATATCACTTTTCGATATTGGGGTGGTCTATATCAATTTAAAAGACAGAATAGATAATTCTTTAGAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCCCATCCTAATTTCTTGCTGATGAGATATTTATTTCTAATTTTTCTATTTTGTCTTTATTTTCAATACTTTCAATCCTATTTTTCTCTTTATTAATAATATAGAACCACCCTATACTATTATACCATATTTTTTGATTTTTCAAAATTCCAATATTTTGTTTTGTGAAATTTTTTCTCCCATTGTCACTTCTCCTGCAAGTACCTTCATTTTTTGAAACTGATCTTCTGTCAGGATAATGGAACGGATTGATGAATTTTCTGGAGCGAGCATTGATAACTGTTTTTCTGCCAGTTCGATTTTTTCTTTTGTTTTCGACCTCATTATATATACCGATTTTTGAAGCTGATAATATCCCTTTTCTATCAATTTTTTCCTAAAAGTCCTATATTCAAATCTCTCAACATCTGTCTGCATAGGAAAATCATACATAAGCAGACCAAAATACTCAATACTCATAGTCCATCACGCTCAATGTCGGAATTATCACTTCTTCATCTTTTACAAAATAATTTCGTATACTATCCAAATAATAGTCTACCGCTTGGAAAAAATCATATTTCTTATTGTTAAATAATACCTTCTGCTGTGCTACAAGAAGTATTTTTTGCCTTATTTCCTTACTTAATTTCACTTCATTCAAAATATCCTTGTACATATAAACAAGATAATCCACCATAGGACGAAAAACCTCTATTATATCATCAGAAAAATTATAGGCATTAAACTGTGACTTATGATGTAATCCTAAACTTGGATGAAATCCTTTTGCTACAATCTTTGATGATATTATAGCTCTTAAAATCATATATCCATAATTAAGTGCAGAATTCACTCCATCTTCATCAAATCTTTTAAAACTATTACTATACAATTCCTGAAAATATATCCTTGAAGCTATTGCTTCCTGATGTTCTGCACTCGCATCATCTTTTTTCAAGTTTTCCTTATATGTTTTCAGTCTTTCAATGGAAATATCACTTTTTTCAAGATACTCTAACAATGCTCTTTGATTTTCAATCTTATTCTCCACTATCCTGCTCCACAATTTTTCCTTTTTCTCTTTTTCCCACTCAATCTGCTCATTTATTCGTAAAGTCACTTGAAAATGATTAAATAATCCCAGCGAATGAATTTCAGGCTGATGTTTCTCGTTGCAAATAATAATCGGAATGTTATTTTCCACCAGCCTCAACTGCAAAATCGCACTAATCTTACAATAGCAGTTTTCAATAACTATCGCAGATATATCATTCAAAGAAATCTTATTTTTCTCATCATTATTGTCTTCATCAACCATTATAAGCTGATTATTCGATATTGACAAATCATCAGCCCTTGTTATGTGAATTATATTGGGCATTTTAATCATACTCCTTATAAATTTCATTCTTATAACGTATCATTCGTATTTTCTATTTTTGTTAAAAGTTCTATTATCAAGTTTTTAATATAATCAGAATTATAACTTTCTAATTCTAAAACAGAAACTTTTTTAGGTTTCATTAATCTTTCAAGTATATCATTATTACCGATAAGTTTAAATTTTTTCTTTAATTCATCATAATCTAAATTCACATCTTTTTTAAATACTTCAAATACACTTGCATAAGTTGAATTATTATAACGTGTACTATATGATAATAAATTAGAAACTCTATCAATTTGTTCTGCAATACTGTAATCAGCAAACGGATTTCTTACAATATAGAAATGTGAAATATAGTTTCTAATACTTTCATTTTCCGGCTTATTAATTTCAGAATTTTCAGACAAATCAATTCCAAATCCATAACATATTTTCTCAAATTTTTTATAAGATTCTTCATCAAAAAATTTATAGTATGCTGTTGTTGTATAAAAGCCATCAGATCCATTACGCTTAGGATAAGCTCTACTTATTCCAGTATTGTAGCCACTTAACTTAATAATTCCTAATTCTCTTAGCCCATTTACAATATAGTGCATATCTCTTTCAAATCTAGCCATTTGAATAGCAAGTTTCCAATTTATATCTATCAAATAACTTTCTATTTTATTCAAATAATTAAATTCTACCAAATCTCTAATTTTTTTGTATTCAGAAACTCTATTATAATCTTTTTCAAATGATTTATAGTTTTTATTTTGTATATTTTTTGCAAAAAAGTCATCATTTTCTTTCAATTTTTTTATATACTTCTCTTTGTATTCTTTAGAATATCCATTTAGTTTATCATTTAGATTTTTCAATATTGCATCAATTTCAGATATTTTATTTTTTCTAATATTTTTACCATCAATATTAAATAAAAATTTTGCATCAGCCATTTTAATATCATTTGAAATTAATCCATAAATTTTATCAAAATTTGGATTTCCAATATTTAAAAATAAATTCTTTTTATAAATATATAATTCATTCTTACGTTCTTTAGGATAATATATTTCTTGAAATTTATTTTCATTCTCTGATTCCATATCTTCTATTAAATTATCTATTTCTTTTTTGTATTTTTTTAAAAAATCAGAATTAAATATTATTCTACACAATATTTTACTCTTTATTTCCTGATCTTTATCTTTTATATACTGATCAACCTTTTTTTTCAAATCCTTTTTATTTATGTTTGATAACTTTCTTTGTTCATCTTGTAATATATTCGATTTTTTATCTATCTCAAATTTAGTTTCATCATCAAAAATTACAATTTTTTCTAATTTTTTCTCTAAAACATCACAACCATTAATATCATCTTTAAATTCAGTTAATATATTATTTTTTATATCCTCATAATAATTATTAAAAATTTCTTTTTTAGTTTGTATTTTAAAATCATCAAAGTCTTTTTCTATCTCTTTCATTTTTTGAATAAATTCTTCTAAATTAAGATTCCAATTTTCAGTTATACATTCATTTCTCAAAGTATTTAATTGCATTATTTCATCTAAAATATCTATAATATTTTGATATTCTGAAGTATTTAACCAAACTGATGTTGCAAAAAATCTATTTCTAATTTTATTTATAACCGCATTACTATTTAACAGTGCAAATATTGAAATTATATATTCAAAATCATCATTTATTACTATAGTTTTATCACTAGTCTTTACAGTTATTCTTTCGTAAGTTTTATTATCATTAATGTCTTTTATTTGTTTCTTAATTTCTTGAATATTCATTTTAAAATCTGAAAAATCAAAAAGTTCCTCATAATTTTTTCTCAAATATCCAATATAACATTCTATTACTTTTTTCTGATATTTTTTAATAGCTTTATTATTACCTTTTGAAGCAGAAATCTGAGCATTTTTATAATAATTTTCTATAATATTTTCATCTATTTCATCAATGTTTCCTAAAGTTTTCTTTAATTCTTGTAAAAATATATTCTTACTTTCATTTTCTTCTAAATCATCTTCTAAAATTAATTTCTTATACAATTCTTTATTCACATATATTAAAGCATTTAATACTATTTTTTCTGTTTCTATAGTATCAAATGGTTCATTCTTAGGATTATTCCTATATAAATTTAATATTTCAGGAAGTACTTTAGAAAAGGATGGTAAATATTTAATATCATTATTATTTTCTTCTGAAATTTTAATATCATTTATTTTAGTAATTATATTTTTTTTATCTTTAAATACTACATCTAAATTTAATGCTTTTGACACTTCTTCATCTGATATTTTTAAATTTTGAATTATATTTATGACTTTATTATAGTCATCTTGCGTTCCTTGTAAATCTCTTTCCTTGCTAATCGCATGTAATATCCTGTTTCTTTCATTTGTTCCTATCTTTGTAAATTTCCTAATAAAATTATTTGTAATGTTATTTTTATTATCTATAAAATCTAAGTCTCTTATTATTTTTATTTTTGAATTTAAAATTTTTTTATCAAGTACGTAATTTTTTTCTCGATCTCCTCCAAAGAAATCTATATTTTCATCATTATTTATATTTTCTCTAGAAAAAATCTTATTTAATTCCATATTGGTAGAAGCAAAAAAAGTAATCAATTCTAAATCCAATTCCTCTTTAGCGTGAAGTCTAGAAAAATCATCAGTATTTACTGTTGTCATATCTATATCATTATGTCTTAATTTCCCTAAATACATAATATGCTCTAACGTATATTGCTTAACTCTTTTTAAAATTTTTTCAGATAATATACTTTCATTTAAAATTTTTTCTATTTCTATTTTTTCCATTTTCTTTAATCTGACTTTTTGTTCATTTACCAATATTTTTTCAATTCTTCCTTTCAAATATCGATATATGATTTTATATAGTTCTTTTTCTTCATCAGATTTCTTTGAAAATTTTTTCGAATCAAAATTAACTTTATAATGTTTTTTAAATATTCCAAAAATTTCTGTATCACAATTTCCTTTTTTTAGTTCTTTTTCTAATTTTTTTATTAATTCATCTATTTTAAATTCTGCTAAAATTTTTTCTATTTTTTCTTTTATACTATTATTTTTTATATTTTCTACAAAAAATTTTACAATTTTATCTTTTTTATTTTCTCTTTCTATTTTAAATTTTTCGTGCTTATCTAATAGTACATAAGATTTTATATATGTTCTATTTCTTCTCTTTTCAAGAAATTCATTATTAACTTTTTTTACTTTTTCAATTCTTTTAGTAATATTCCAAAATTCTAACTCTTTTATAACAAAATCAGCTATATCTTCTACTGTTAAATCTACATTTATATTTAAAATTTTTTCAACAAGCATTTTTTTATTTTTAGATTTCTTTTTATCACCACCAACATTAAGATAAAATTTTACAAAACCCAGAATTTCTAAATTACTTTTTATTTTTTCTCTTATTTCCATAAAATTAGTCAAAATAACATCTATTTTATCATCTTTCAATAATTTTTCTCTTAAATGTTCTTCATAATATCGATTTTCAAATACTTTTTCTGTTTCATTTTCAATTATTTTTTCTATAATCTTATATAAACTCATGTTAATATTTTTAAAAATTTCGTAAATTGATTTTTTTGTTTCTAATTCATCATTTTCTATTATTCTTAATATTATTGAACAATCATTTAGTGTTTTATTAGTATACTCATCTCTGATATCTATCTCTATTTCTTCTTCATTCTCTTGTCTCTTTATTTCTATTTTTTTATCATCTTTAGTTATTCCTTGCCTAATTGCTTCATCTATTATTTTCTTTTTTGTAATCCCCAATGCTTTCAATTTCTCAGATTTTCCATATGCTTCTATATATAATACAACTTCTTCTGTTTCCAAAAAATCATCATTATTTTCTATTCTTATGATTCCTTCTTTACCTTTCAACTTAAATAGAATATTTCCTGCATGAAATTTTCTTGTAAATTCTTTAAGAATATTATCATTTTTTTTGTAATTAATATATTTTCTAATAAATTTATTATTATCAATTTTTTCTTTATTATTATTTTCATTAATATTTAAAATGTATTTGTTTCCATCATAGTTCCTTTTAACTTTTACTTTCCGTTTTATTTTAAAATCTTTTTTATCACGAACTTCATACCATCTCTTATGTCCAAATAAATTTCCCATTCCAATCTCCTCGTTTCTACTTTAATCTAATAAAATATTTTTAAATTAAATCAATTTTACATCTTTCTAATCAAAAATACAATTTTCCATTTTTAGTATACCACATCAATATTAAATCTCAAAAAAATAAGGAGCCGTCAAACATAGCTCCCTACTTCTATTTACTCATAATCCCCATCTATCCTTACTTTTCGTAAAATCAATCCTTCTTTCGCCTTTAGATCCAACTTAATTTTCCCATTTGAACCTGTTCTAAATGTTCTGCCTTCTGTTACCAAATCAATAAATCTTTCATCCTGATAATTTGTTTCAAATTCCACATTTTCCCAGCTGTTAAACGAATTATTTATTACAACAATAATTAAATGATCCTCGATTACTCTTTCATACACAATTATTT

Example 3: Further Evaluation of Cpf1 and Associated Components

Applicants carried out sequence alignments with Cas-Cpf1 orthologs andcompared the domain structure and organization (FIG. 38A-N). An overviewof Cpf1 loci alignment in shown in FIG. 39 .

The sequences of Cpf1 loci in various orthologs are listed below:

>KKP36646_(modified) hypothetical protein UR27_C0015G0004 [Peregrinibacteriabacterium GW2011_GWA2_33_10] (SEQ ID NO: 68)MSNFFKNFTNLYELSKTLRFELKPVGDTLTNMIKDHLEYDEKLQTFLKDQNIDDAYQALKPQFDEIHEEFITDSLESKKAKEIDFSEYLDLFQEKKELNDSEKKLRNKIGETFNKAGEKWKKEKYPQYEWKKGSKIANGADILSCQDMLQFIKYKNPEDEKIKNYIDDTLKGFFTYFGGFNQNRANYYETKKEASTAVATRIVHENLPKFCDNVIQFKHIIKRKKDGTVEKTERKTEYLNAYQYLKNNNKITQIKDAETEKMIESTPIAEKIFDVYYFSSCLSQKQIEEYNRIIGHYNLLINLYNQAKRSEGKHLSANEKKYKDLPKFKTLYKQIGCGKKKDLFYTIKCDTEEEANKSRNEGKESHSVEEIINKAQEAINKYFKSNNDCENINTVPDFINYILTKENYEGVYWSKAAMNTISDKYFANYHDLQDRLKEAKVFQKADKKSEDDIKIPEAIELSGLFGVLDSLADWQTTLFKSSILSNEDKLKIITDSQTPSEALLKMIFNDIEKNMESFLKETNDIITLKKYKGNKEGTEKIKQWFDYTLAINRMLKYFLVKENKIKGNSLDTNISEALKTLIYSDDAEWFKWYDALRNYLTQKPQDEAKENKLKLNFDNPSLAGGWDVNKECSNFCVILKDKNEKKYLAIMKKGENTLFQKEWTEGRGKNLTKKSNPLFEINNCEILSKMEYDFWADVSKMIPKCSTQLKAVVNHFKQSDNEFIFPIGYKVTSGEKFREECKISKQDFELNNKVFNKNELSVTAMRYDLSSTQEKQYIKAFQKEYWELLFKQEKRDTKLTNNEIFNEWINFCNKKYSELLSWERKYKDALTNWINFCKYFLSKYPKTTLFNYSFKESENYNSLDEFYRDVDICSYKLNINTTINKSILDRLVEEGKLYLFEIKNQDSNDGKSIGHKNNLHTIYWNAIFENFDNRPKLNGEAEIFYRKAISKDKLGIVKGKKTKNGTEIIKNYRFSKEKFILHVPITLNFCSNNEYVNDIVNTKFYNFSNLHFLGIDRGEKHLAYYSLVNKNGEIVDQGTLNLPFTDKDGNQRSIKKEKYFYNKQEDKWEAKEVDCWNYNDLLDAMASNRDMARKNWQRIGTIKEAKNGYVSLVIRKIADLAVNNERPAFIVLEDLNTGFKRSRQKIDKSVYQKFELALAKKLNFLVDKNAKRDEIGSPTKALQLTPPVNNYGDIENKKQAGIMLYTRANYTSQTDPATGWRKTIYLKAGPEETTYKKDGKIKNKSVKDQIIETFTDIGFDGKDYYFEYDKGEFVDEKTGEIKPKKWRLYSGENGKSLDRFRGEREKDKYEWKIDKIDIVKILDDLFVNFDKNISLLKQLKEGVELTRNNEHGTGESLRFAINLIQQIRNTGNNERDNDFILSPVRDENGKHFDSREYWDKETKGEKISMPSSGDANGAFNIARKGIIMNAHILANSDSKDLSLFVSDEEWDLHLNNKTEWKKQLNIFSSRKAMAKRKK>KKR91555_(modified) hypothetical protein UU43_C0004G0003 [Parcubacteria(Falkowbacteria) bacterium GW2011_GWA2_41_14] (SEQ ID NO: 69)MLFFMSTDITNKPREKGVFDNFTNLYEFSKTLTFGLIPLKWDDNKKMIVEDEDFSVLRKYGVIEEDKRIAESIKIAKFYLNILHRELIGKVLGSLKFEKKNLENYDRLLGEIEKNNKNENISEDKKKEIRKNFKKELSIAQDILLKKVGEVFESNGSGILSSKNCLDELTKRFTRQEVDKLRRENKDIGVEYPDVAYREKDGKEETKSFFAMDVGYLDDFHKNRKQLYSVKGKKNSLGRRILDNFEIFCKNKKLYEKYKNLDIDFSEIERNFNLTLEKVFDFDNYNERLTQEGLDEYAKILGGESNKQERTANIHGLNQIINLYIQKKQSEQKAEQKETGKKKIKFNKKDYPTFTCLQKQILSQVFRKEIIIESDRDLIRELKFFVEESKEKVDKARGIIEFLLNREENDIDLAMVYLPKSKINSFVYKVFKEPQDFLSVFQDGASNLDFVSFDKIKTHLENNKLTYKIFFKTIIKENHDFESFLILLQQEIDLLIDGGETVTLGGKKESITSLDEKKNRLKEKLGWFEGKVRENEKMKDEEEGEFCSTVLAYSQAVLNITKRAEIFWLNEKQDAKVGEDNKDMIFYKKFDEFADDGFAPFFYFDKFGNYLKRRSRNTTKEIKLHFGNDDLLEGWDMNKEPEYWSFILRDRNQYYLGIGKKDGEIFHKKLGNSVEAVKEAYELENEADFYEKIDYKQLNIDRFEGIAFPKKTKTEEAFRQVCKKRADEFLGGDTYEFKILLAIKKEYDDFKARRQKEKDWDSKFSKEKMSKLIEYYITCLGKRDDWKRFNLNFRQPKEYEDRSDFVRHIQRQAYWIDPRKVSKDYVDKKVAEGEMFLFKVHNKDFYDFERKSEDKKNHTANLFTQYLLELFSCENIKNIKSKDLIESIFELDGKAEIRFRPKTDDVKLKIYQKKGKDVTYADKRDGNKEKEVIQHRRFAKDALTLHLKIRLNFGKHVNLFDFNKLVNTELFAKVPVKILGMDRGENNLIYYCFLDEHGEIENGKCGSLNRVGEQIITLEDDKKVKEPVDYFQLLVDREGQRDWEQKNWQKMTRIKDLKKAYLGNVVSWISKEMLSGIKEGVVTIGVLEDLNSNFKRTRFFRERQVYQGFEKALVNKLGYLVDKKYDNYRNVYQFAPIVDSVEEMEKNKQIGTLVYVPASYTSKICPHPKCGWRERLYMKNSASKEKIVGLLKSDOKISYDQKNDRFYFEYQWEQEHKSDGKKKKYSGVDKVFSNVSRMRWDVEQKKSIDFVDGTDGSITNKLKSLLKGKGIELDNINQQIVNQQKELGVEFFQSIIFYFNLIMQIRNYDKEKSGSEADYIQCPSCLFDSRKPEMNGKLSAITNGDANGAYNIARKGFMQLCRIRENPQEPMKLITNREWDEAVREWDIYSAAQKIPVLSEEN>KDN25524_(modified) hypothetical protein MBO_03467 [Moraxella bovoculi 237](SEQ ID NO: 70) MLFQDFTHLYPLSKTVRFELKPIDRTLEHIHAKNFLSQDETMADMHQKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDDELQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDGKELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYSDEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTASGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSPKIQGINELINSHHNQHCHKSERIAKLRPLHKQILSDGMSVSFLPSKFADDSEMCQAVNEFYRHYADVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKSIYQKMIYKYLEVRKQFPKVFFSKEAIAINYHPSKELVEIKDKGRQRSDDERLKLYRFILECLKIHPKYDKKFEGAIGDIQLFKKDKKGREVPISEKDLFDKINGIFSSKPKLEMEDFFIGEFKRYNPSQDLVDQYNIYKKIDSNDNRKKENFYNNHPKFKKDLVRYYYESMCKHEEWEESFEFSKKLQDIGCYVDVNELFTEIETRRLNYKISFCNINADYIDELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQCSLNDITTASANGTQMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQISQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADKDYFEFHIDYAKFTDKAKNSRQIWTICSHGDKRYVYDKTANQNKGAAKGINVNDELKSLFARHHINEKQPNLVMDICQNNDKEFHKSLMYLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR>KKT48220_(modified) hypothetical protein UW39_C0001G0044 [Parcubacteriabacterium GW2011_GWC2_44_17] (SEQ ID NO: 71)MENIFDQFIGKYSLSKTLRFELKPVGKTEDFLKINKVFEKDQTIDDSYNQAKFYFDSLHQKFIDAALASDKTSELSFQNFADVLEKQNKIILDKKREMGALRKRDKNAVGIDRLQKEINDAEDIIQKEKEKIYKDVRTLFDNEAESWKTYYQEREVDGKKITFSKADLKQKGADFLTAAGILKVLKYEFPEEKEKEFQAKNQPSLFVEEKENPGQKRYIFDSFDKFAGYLTKFQQTKKNLYAADGTSTAVATRIADNFIIFHQNTKVFRDKYKNNHTDLGFDEENIFEIERYKNCLLQREIEHIKNENSYNKIIGRINKKIKEYRDQKAKDTKLTKSDFPFFKNLDKQILGEVEKEKQLIEKTREKTEEDVLIERFKEFIENNEERFTAAKKLMNAFCNGEFESEYEGIYLKNKAINTISRRWFVSDRDFELKLPQQKSKNKSEKNEPKVKKFISIAEIKNAVEELDGDIFKAVFYDKKIIAQGGSKLEQFLVIWKYEFEYLFRDIERENGEKLLGYDSCLKIAKQLGIFPQEKEAREKATAVIKNYADAGLGIFQMMKYFSLDDKDRKNTPGQLSTNFYAEYDGYYKDFEFIKYYNEFRNFITKKPFDEDKIKLNFENGALLKGWDENKEYDFMGVILKKEGRLYLGIMHKNHRKLFQSMGNAKGDNANRYQKMIYKQIADASKDVPRLLLTSKKAMEKFKPSQEILRIKKEKTFKRESKNFSLRDLHALIEYYRNCIPQYSNWSFYDFQFQDTGKYQNIKEFTDDVQKYGYKISFRDIDDEYINQALNEGKMYLFEVVNKDIYNTKNGSKNLHTLYFEHILSAENLNDPVFKLSGMAEIFQRQPSVNEREKITTQKNQCILDKGDRAYKYRRYTEKKIMFHMSLVLNTGKGEIKQVQFNKIINQRISSSDNEMRVNVIGIDRGEKNLLYYSVVKQNGEIIEQASLNEINGVNYRDKLIEREKERLKNRQSWKPVVKIKDLKKGYISHVIHKICQLIEKYSAIVVLEDLNMRFKQIRGGIERSVYQQFEKALIDKLGYLVFKDNRDLRAPGGVLNGYQLSAPFVSFEKMRKQTGILFYTQAEYTSKTDPITGFRKNVYISNSASLDKIKEAVKKFDAIGWDGKEQSYFFKYNPYNLADEKYKNSTVSKEWAIFASAPRIRRQKGEDGYWKYDRVKVNEEFEKLLKVWNFVNPKATDIKQEIIKKEKAGDLQGEKELDGRLRNFWHSFIYLFNLVLELRNSFSLQIKIKAGEVIAVDEGVDFIASPVKPFFTTPNPYIPSNLCWLAVENADANGAYNIARKGVMILKKIREHAKKDPEFKKLPNLFISNAEWDEAARDWGKYAGTTALNLDH>WP_031492824_(modified) hypothetical protein [Succinivibrio dextrinosolvens](SEQ ID NO: 72) MSSLTKFTNKYSKQLTIKNELIPVGKTLENIKENGLIDGDEQLNENYQKAKIIVDDFLRDFINKALNNTQIGNWRELADALNKEDEDNIEKLQDKIRGIIVSKFETFDLFSSYSIKKDEKIIDDDNDVEEEELDLGKKTSSFKYIFKKNLFKLVLPSYLKTTNQDKLKIISSFDNFSTYFRGFFENRKNIFTKKPISTSIAYRIVHDNFPKFLDNIRCFNVWQTECPQLIVKADNYLKSKNVIAKDKSLANYFTVGAYDYFLSQNGIDFYNNIIGGLPAFAGREKIQGLNEFINQECQKDSELKSKLKNRHAFKMAVLFKQILSDREKSFVIDEFESDAQVIDAVKNFYAEQCKDNNVIFNLLNLIKNIAFLSDDELDGIFIEGKYLSSVSQKLYSDWSKLRNDIEDSANSKQGNKELAKKIKTNKGDVEKAISKYEFSLSELNSIVHDNTKFSDLLSCTLHKVASEKLVKVNEGDWPKHLKNNEEKQKIKEPLDALLEIYNTLLIFNCKSFNKNGNFYVDYDRCINELSSVVYLYNKTRNYCTKKPYNTDKFKLNFNSPQLGEGFSKSKENDCLTLLFKKDDNYYVGIIRKGAKINFDDTQAIADNTDNCIFKMNYFLLKDAKKFIPKCSIQLKEVKAHFKKSEDDYILSDKEKFASPLVIKKSTFLLATAHVKGKKGNIKKFQKEYSKENPTEYRNSLNEWIAFCKEFLKTYKAATIFDITTLKKAEEYADIVEFYKDVDNLCYKLEFCPIKTSFIENLIDNGDLYLFRINNKDFSSKSTGTKNLHTLYLQAIFDERNLNNPTIMLNGGAELFYRKESIEQKNRITHKAGSILVNKVCKDGTSLDDKIRNEIYQYENKFIDTLSDEAKKVLPNVIKKEATHDITKDKRFTSDKFFFHCPLTINYKEGDTKQFNNEVLSFLRGNPDINIIGIDRGERNLIYVTVINQKGEILDSVSFNTVTNKSSKIEQTVDYEEKLAVREKERIEAKRSWDSISKIATLKEGYLSAIVHEICLLMIKHNAIVVLENLNAGFKRIRGGLSEKSVYQKFEKMLINKLNYFVSKKESDWNKPSGLLNGLQLSDQFESFEKLGIQSGFIFYVPAAYTSKIDPTTGFANVLNLSKVRNVDAIKSFFSNFNEISYSKKEALFKFSFDLDSLSKKGFSSFVKFSKSKWNVYTFGERIIKPKNKQGYREDKRINLTFEMKKLLNEYKVSFDLENNLIPNLTSANLKDTFWKELFFIFKTTLQLRNSVTNGKEDVLISPVKNAKGEFFVSGTHNKTLPQDCDANGAYHIALKGLMILERNNLVREEKDTKKIMAISNVDWFEYVQKRRGVL>KKT50231_(modified) hypothetical protein UW40_C0007G0006 [Parcubacteriabacterium GW2011_GWF2_44_17] (SEQ ID NO: 73)MKPVGKTEDFLKINKVFEKDQTIDDSYNQAKFYFDSLHQKFIDAALASDKTSELSFQNFADVLEKQNKIILDKKREMGALRKRDKNAVGIDRLQKEINDAEDIIQKEKEKIYKDVRTLFDNEAESWKTYYQEREVDGKKITFSKADLKQKGADFLTAAGILKVLKYEFPEEKEKEFQAKNQPSLFVEEKENPGQKRYIFDSFDKFAGYLTKFQQTKKNLYAADGTSTAVATRIADNFIIFHQNTKVFRDKYKNNHTDLGFDEENIFEIERYKNCLLQREIEHIKNENSYNKIIGRINKKIKEYRDQKAKDTKLTKSDFPFFKNLDKQILGEVEKEKQLIEKTREKTEEDVLIERFKEFIENNEERFTAAKKLMNAFCNGEFESEYEGIYLKNKAINTISRRWFVSDRDFELKLPQQKSKNKSEKNEPKVKKFISIAEIKNAVEELDGDIFKAVFYDKKIIAQGGSKLEQFLVIWKYEFEYLFRDIERENGEKLLGYDSCLKIAKQLGIFPQEKEAREKATAVIKNYADAGLGIFQMMKYFSLDDKDRKNTPGQLSTNFYAEYDGYYKDFEFIKYYNEFRNFITKKPFDEDKIKLNFENGALLKGWDENKEYDFMGVILKKEGRLYLGIMHKNHRKLFQSMGNAKGDNANRYQKMIYKQIADASKDVPRLLLTSKKAMEKFKPSQEILRIKKEKTFKRESKNFSLRDLHALIEYYRNCIPQYSNWSFYDFQFQDTGKYQNIKEFTDDVQKYGYKISFRDIDDEYINQALNEGKMYLFEVVNKDIYNTKNGSKNLHTLYFEHILSAENLNDPVFKLSGMAEIFQRQPSVNEREKITTQKNQCILDKGDRAYKYRRYTEKKIMFHMSLVLNTGKGEIKQVQFNKIINQRISSSDNEMRVNVIGIDRGEKNLLYYSVVKQNGEIIEQASLNEINGVNYRDKLIEREKERLKMQSWKPVVKIKDLKKGYISHVIHKICQLIEKYSAIVVLEDLNMRFKQIRGGIERSVYQQFEKALIDKLGYLVFKDNRDLRAPGGVLNGYQLSAPFVSFEKMRKQTULFYTQAEYTSKTDPITGFRKNVYISNSASLDKIKEAVKKFDAIGWDGKEQSYFFKYNPYNLADEKYKNSTVSKEWAIFASAPRIRRQKGEDGYWKYDRVKVNEEFEKLLKVWNFVNPKATDIKQEIIKKEKAGDLQGEKELDGRLRNFWHSFIYLFNLVLELRNSFSLQIKIKAGEVIAVDEGVDFIASPVKPFFTTPNPYIPSNLCWLAVENADANGAYNIARKGVMILKKIREHAKKDPEFKKLPNLFISNAEWDEAARDWGKYAGTTALNLDH>WP_004356401_(modified) hypothetical protein [Prevotella disiens](SEQ ID NO: 74) MENYQEFTNLFQLNKTLRFELKPIGKTCELLEEGKIFASGSFLEKDKVRADNVSYVKKEIDKKHKIFIEETLSSFSISNDLLKQYFDCYNELKAFKKDCKSDEEEVKKTALRNKCTSIQRAMREAISQAFLKSPQKKLLAIKNLIENVFKADENVQHFSEFTSYFSGFETNRENFYSDEEKSTSIAYRLVHDNLPIFIKNIYIFEKLKEQFDAKTLSEIFENYKLYVAGSSLDEVFSLEYFNNTLTQKGIDNYNAVIGKIVKEDKQEIQGLNEHINLYNQKHKDRRLPFFISLKKQILSDREALSWLPDMFKNDSEVIKALKGFYIEDGFENNVLTPLATLLSSLDKYNLNGIFIRNNEALSSLSQNVYRNFSIDEAIDANAELQTFNNYELIANALRAKIKKETKQGRKSFEKYEEYIDKKVKAIDSLSIQEINELVENYVSEFNSNSGNMPRKVEDYFSLMRKGDFGSNDLIENIKTKLSAAEKLLGTKYQETAKDIFKKDENSKLIKELLDATKQFQHFIKPLLGTGEEADRDLVFYGDFLPLYEKFEELTLLYNKVRNRLTQKPYSKDKIRLCFNKPKLMTGWVDSKTEKSDNGTQYGGYLFRKKNEIGEYDYFLGISSKAQLFRKNEAVIGDYERLDYYQPKANTIYGSAYEGENSYKEDKKRLNKVIIAYIEQIKQTNIKKSIIESISKYPNISDDDKVTPSSLLEKIKKVSIDSYNGILSFKSFQSVNKEVIDNLLKTISPLKNKAEFLDLINKDYQIFTEVQAVIDEICKQKTFIYFPISNVELEKEMGDKDKPLCLFQISNKDLSFAKTFSANLRKKRGAENLHTMLFKALMEGNQDNLDLGSGAIFYRAKSLDGNKPTHPANEAIKCRNVANKDKVSLFTYDIYKNRRYMENKFLFHLSIVQNYKAANDSAQLNSSATEYIRKADDLHIIGIDRGERNLLYYSVIDMKGNIVEQDSLNIIRNNDLETDYHDLLDKREKERKANRQNWEAVEGIKDLKKGYLSQAVHQIAQLMLKYNAIIALEDLGQMFVTRGQKIEKAVYQQFEKSLVDKLSYLVDKKRPYNELGGILKAYQLASSITKNNSDKQNGFLFYVPAWNTSKIDPVTGFTDLLRPKAMTIKEAQDFFGAFDNISYNDKGYFEFETNYDKFKIRMKSAQTRWTICTFGNRIKRKKDKNYWNYEEVELTEEFKKLFKDSNIDYENCNLKEEIQNKDNRKFFDDLIKLLQLTLQMRNSDDKGNDYIISPVANAEGQFFDSRNGDKKLPLDADANGAYNIARKGLWNIRQIKQTKNDKKLNLSISSTEWL DFVREKPYLK>CCB70584_(modified) Protein of unknown function [Flavobacteriumbranchiophilum FL-15] (SEQ ID NO: 75)MTNKFTNQYSLSKTLRFELIPQGKTLEFIQEKGLLSQDKQRAESYQEMKKTIDKFIAKYFIDLALSNAKLTHLETYLELYNKSAETKKEQKFKDDLKKVQDNLRKEIVKSFSDGDAKSIFAILDKKELITVELEKWFENNEQKDIYFDEKFKTFTTYFTGFHQNRKNMYSVEPNSTAIAYRLIHENLPKFLENAKAFEKIKQVESLQVNFRELMGEFGDEGLIFVNELEEMFQINYYNDVLSQNGITIYNSIISGFTKNDIKYKGLNEYINNYNQTKDKKDRLPKLKQLYKQILSDRISLSFLPDAFTDGKQVLKAIFDFYKINLLSYTIEGQEESQNLLLLIRQTIENLSSFDTQKIYLKNDTHLTTISQQVFGDFSVFSTALNYWYETKVNPKFETEYSKANEKKREILDKAKAVFTKQDYFSIAFLQEVLSEYILTLDHTSDIVKKHSSNCIADYFKNEEFVAKKENETDKTFDFIANITAKYQCIQGILENADQYEDELKQDQKLIDNLKFFLDAILELLHFIKPLHLKSESITEKDTAFYDVFENYYEALSLLTPLYNMVRNYVTQKPYSTEKIKLNFENAQLLNGWDANKEGDYLTTILKKDGNYFLAIMDKKHNKAFQKFPEGKENYEKMVYKLLPGVNKMLPKVFFSNKNIAYFNPSKELLENYKKETIAKKGDTFNLEHCHTLIDFFKDSLNKREDWKYFDFQFSETKSYQDLSGFYREVEHQGYKINFKNIDSEYIDGLVNEGKLFLFQIYSKDFSPFSKGKPNMHTLYWKALFEEQNLQNVIYKLNGQAEIFFRKASIKPKNIILHKKKIKIAKKHFIDKKTKTSEIVPVQTIKNLNMYYQGKISEKELTQDDLRYIDNFSIFNEKNKTIDIIKDKRFTVDKFQFHVPITMNFKATGGSYINQTVLEYLQNNPEVKIIGLDRGERHLVYLTLIDQQGNILKQESLNTITDSKISTPYHKLLDNKENERDLARKNWGTVENIKELKEGYISQVVHKIATLMLEENAIVVMEDLNFGFKRGRFKVEKQIYQKLEKMLIDKLNYLVLKDKQPQELGGLYNALQLTNKFESFQKMGKQSGFLFYVPAWNTSKIDPTTGFVNYFYTKYENVDKAKAFFEKFEAIRFNAEKKYFEFEVKKYSDFNPKAEGTQQAWTICTYGERIETKRQKDQNNKFVSTPINLTEKIEDFLGKNQIVYGDGNCIKSQIASKDDKAFFETLLYWFKMTLQMRNSETRTDIDYLISPVMNDNGTFYNSRDYEKLENPTLPKDADANGAYHIAKKGLMLLNKIDQADLTKKVDLSISNR DWLQFVQKNK>WP_005398606_(modified) hypothetical protein [Helcococcus kunzii](SEQ ID NO: 76) MFEKLSNIVSISKTIRFKLIPVGKTLENIEKLGKLEKDFERSDFYPILKNISDDYYRQYIKEKLSDLNLDWQKLYDAHELLDSSKKESQKNLEMIQAQYRKVLFNILSGELDKSGEKNSKDLIKNNKALYGKLFKKQFILEVLPDFVNNNDSYSEEDLEGLNLYSKFTTRLKNFWETRKNVFTDKDIVTAIPFRAVNENFGFYYDNIKIFNKNIEYLENKIPNLENELKEADILDDNRSVKDYFTPNGFNYVITQDGIDVYQAIRGGFTKENGEKVQGINEILNLTQQQLRRKPETKNVKLGVLTKLRKQILEYSESTSFLIDQIEDDNDLVDRINKFNVSFFESTEVSPSLFEQIERLYNALKSIKKEEVYIDARNTQKFSQMLFGQWDVIRRGYTVKITEGSKEEKKKYKEYLELDETSKAKRYLNIREIEELVNLVEGFEEVDVFSVLLEKFKMNNIERSEFEAPIYGSPIKLEAIKEYLEKHLEEYHKWKLLLIGNDDLDTDETFYPLLNEVISDYYTIPLYNLTRNYLTRKHSDKDKIKVNFDFPTLADGWSESKISDNRSIILRKGGYYYLGILIDNKLLINKKNKSKKIYEILIYNQIPEFSKSIPNYPFTKKVKEHFKNNVSDFQLIDGYVSPLIITKEIYDIKKEKKYKKDFYKDNNTNKNYLYTIYKWIEFCKQFLYKYKGPNKESYKEMYDFSTLKDTSLYVNLNDFYADVNSCAYRVLFNKIDENTIDNAVEDGKLLLFQIYNKDFSPESKGKKNLHTLYWLSMFSEENLRTRKLKLNGQAEIFYRKKLEKKPIIHKEGSILLNKIDKEGNTIPENIYEEECYRYLNKKIGREDLSDEAIALFNKDVLKYKEARFDIIKDRRYSESQFFFHVPITFNWDIKTNKNVNQIVQGMIKDGEIKHIIGIDRGERHLLYYSVIDLEGNIVEQGSLNTLEQNRFDNSTVKVDYQNKLRTREEDRDRARKNWTNINKIKELKDGYLSHVVHKLSRLIIKYEAIVIMENLNQGFKRGRFKVERQVYQKFELALMNKLSALSFKEKYDERKNLEPSGILNPIQACYPVDAYQELQGQNGIVFYLPAAYTSVIDPVTGFTNLFRLKSINSSKYEEFIKKFKNIYFDNEEEDFKFIFNYKDFAKANLVILNNIKSKDWKISTRGERISYNSKKKEYFYVQPTEFLINKLKELNIDYENIDIIPLIDNLEEKAKRKILKALFDTFKYSVQLRNYDFENDYIISPTADDNGNYYNSNEIDIDKTNLPNNGDANGAFNIARKGLLLKDRIVNSNESKVDLKIKNEDWINFIIS >WP_021736722_(modified) CRISPR-associated protein Cpf1 , subtype PREFRAN[Acidaminococcus sp. BV3L6] (SEQ ID NO: 77)MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEIVIEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSEIDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN>WP_004339290_(modified) hypothetical protein [Francisella tularensis](SEQ ID NO: 78) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISKYINDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQVAPKNLDNPSKKEQDLIAKKTEKAKYLSLETIKLALEEFNKEIRDIDKQCRFEEILSNFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEEDVKAIKDLLDQTNNLLHRLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLASGWDKNKESANTAILFIKDDKYYLGIMDKKHNKIFSDKAIEENKGEGYKKIVYKQIADASKDIQNLMIIDGKTVCKKGRKDRNGVNRQLLSLKRKHLPENIYRIKETKSYLKNEARFSRKDLYDFIDYYKDRLDYYDFEFELKPSNEYSDFNDFTNHIGSQGYKLTFENISQDYINSLVNEGKLYLFQIYSKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKETIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDNFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLDRIKNNQEGKKLNLVIKNEEYFEFVQNRNN>WP_022501477_(modified) hypothetical protein [Eubacterium sp. CAG: 76](SEQ ID NO: 79) MNKAADNYTGGNYDEFIALSKVQKTLRNELKPTPFTAEHIKQRGIISEDEYRAQQSLELKKIADEYYRNYITHKLNDINNLDFYNLFDAIEEKYKKNDKDNRDKLDLVEKSKRGEIAKMLSADDNFKSMFEAKLITKLLPDYVERNYTGEDKEKALETLALFKGFTTYFKGYFKTRKNMFSGEGGASSICHRIVNVNASIFYDNLKTFMRIQEKAGDEIALIEEELTEKLDGWRLEHIFSRDYYNEVLAQKGIDYYNQICGDINKHMNLYCQQNKFKANIFKMMKIQKQIMGISEKAFEIPPMYQNDEEVYASFNEFISRLEEVKLTDRLINILQNINIYNTAKIYINARYYTNVSSYVYGGWGVIDSAIERYLYNTIAGKGQSKVKKIENAKKDNKFMSVKELDSIVAEYEPDYFNAPYIDDDDNAVKAFGGQGVLGYFNKMSELLADVSLYTIDYNSDDSLIENKESALRIKKQLDDIMSLYHWLQTFIIDEVVEKDNAFYAELEDICCELENVVTLYDRIRNYVTKKPYSTQKFKLNFASPTLAAGWSRSKEFDNNAIILLRNNKYYIAIFNVNNKPDKQIIKGSEEQRLSTDYKKMVYNLLPGPNKMLPKVFIKSDTGKRDYNPSSYILEGYEKNRHIKSSGNFDINYCHDLIDYYKACINKEEPEWKNYGFKFKETNQYNDIGQFYKDVEKQGYSISWAYISEEDINKLDEEGKIYLFEIYNKDLSAHSTGRDNLHTMYLKNIFSEDNLKNICIELNGEAELFYRKSSMKSNITEIKKDTILVNKTYINETGVRVSLSDEDYMKVYNYYNNNYVIDTENDKNLIDIIEKIGHRKSKIDIVKDKRYTEDKYFLYLPITINYGIEDENVNSKIIEYIAKQDNMNVIGIDRGERNLIYISVIDNKGNIIEQKSFNLVNNYDYKNKLKNMEKTRDNARKNWQEIGKIKDVKSGYLSGVISKIARMVIDYNAIIVMEDLNKGFKRGRFKVERQVYQKFENMLISKLNYLVFKERKADENGGILRGYQLTYIPKSIKNVGKQCGCIFYVPAAYTSKIDPATGFINIFDFKKYSGSGINAKVKDKKEFLMSMNSIRYINECSEEYEKIGHRELFAFSFDYNNFKTYNVSSPVNEWTAYTYGERIKKLYKDGRWLRSEVLNLTENLIKLMEQYNIEYKDGHDIREDISHMDETRNADFICSLFEELKYTVQLRNSKSEAEDENYDRLVSPILNSSNGFYDSSDYMENENNTTHTMPKDADANGAYCIALKGLYEINKIKQNWSDDKKFKENELYINVTEWLDYIQNRRFE>WP_014550095_(modified) hypothetical protein [Francisella tularensis](SEQ ID NO: 80) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQVAPKNLDNPSKKEQDLIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHRLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGNPQKGYEKFEFNIEDCRKFIDFYKESISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKKSIPKKITHPAKEAIANKNKDNPKKESFFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVREIAKLVIEHNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGITYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSILNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLDRIKNNQEGKKLNLVIKNEEYFEFVQNRNN>WP_003034647_(modified) hypothetical protein [Francisella tularensis](SEQ ID NO: 81)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSDDIPTSITYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQVAPKNLDNPSKKEQDLIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISLKYQNQGKKDLLQASAEEDVKAIKDLLDQTNNLLHRLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGNPQKGYEKFEFNIEDCRKFIDFYKESISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEHNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLDRIKNNQEGKKLNLVIKNEEYFEFVQNRN N>FnCpf1 Francisella tularensis subsp. novicida U112, complete genome(SEQ ID NO: 82) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGULDDEKRAKDYKKAKQIIDKYHQFFIEELSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKEEPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN>KKQ38174_(modified) hypothetical protein US54_C0016G0015 [Microgenomates(Roizmanbacteria) bacterium GW2011_GWA2_37_7] (SEQ ID NO: 83)MKSFDSFTNLYSLSKTLKFEMRPVGNTQKMLDNAGVFEKDKLIQKKYGKTKPYFDRLHREFIEEALTGVELIGLDENFRTLVDWQKDKKNNVAMKAYENSLQRLRTEIGKIFNLKAEDWVKNKYPILGLKNKNTDILFEEAVFGILKARYGEEKDTFIEVEEIDKTGKSKINQISIFDSWKGFTGYFKKFFETRKNFYKNDGTSTAIATRIIDQNLKRFIDNLSIVESVRQKVDLAETEKSFSISLSQFFSIDFYNKCLLQDGIDYYNKIIGGETLKNGEKLIGLNELINQYRQNNKDQKIPFFKLLDKQILSEKILFLDEIKNDTELLEALSQFAKTAEEKTKIVKKLFADFVENNSKYDLAQIYISQEAFNTISNKWTSETETFAKYLFEAMKSGKLAKYEKKDNSYKFPDFIALSQMKSALLSISLEGHFWKEKYYKISKFQEKTNWEQFLALFLYEFNSLFSDKINTKDGETKQVGYYLFAKDLHNLILSEQIDIPKDSKVTIKDFADSVLTIYQMAKYFAVEKKRAWLAEYIELDSFYTQPDTGYLQFYDNAYEDIVQVYNKLRNYLTKKPYSEEKWKLNFENSTLANGWDKNKESDNSAVILQKGGKYYLGLITKGHNKIFDDRFQEKFIVGIEGGKYEKIVYKFFPDQAKMFPKVCFSAKGLEFFRPSEEILRIYNNAEFKKGETYSIDSMQKLIDFYKDCLTKYEGWACYTFRHLKPTEEYQNNIGEFFRDVAEDGYRIDFQGISDQYIHEKNEKGELHLFEIHNKDWNLDKARDGKSKTTQKNLHTLYFESLFSNDNVVQNFPIKLNGQAEIFYRPKTEKDKLESKKDKKGNKVIDHKRYSENKIFFHVPLTLNRTKNDSYRFNAQINNFLANNKDINIIGVDRGEKHLVYYSVITQASDILESGSLNELNGVNYAEKLGKKAENREQARRDWQDVQGIKDLKKGYISQVVRKLADLAIKHNAIIILEDLNMRFKQVRGGIEKSIYQQLEKALIDKLSFLVDKGEKNPEQAGHLLKAYQLSAPFETFQKMGKQTGIIFYTQASYTSKSDPVTGWRPHLYLKYFSAKKAKDDIAKFTKIEFVNDRFELTYDIKDFQQAKEYPNKTVWKVCSNVERFRWDKNLNQNKGGYTHYTNITENIQELFTKYGIDITKDLLTQISTIDEKQNTSFFRDFIFYFNLICQIRNTDDSEIAKKNGKDDFILSPVEPFFDSRKDNGNKLPENGDDNGAYNIARKGIVILNKISQYSEKNENCEKMKWGDLYVSNIDWDNFVTQANARH>WP_022097749_(modified) hypothetical protein [Eubacterium eligens CAG: 72](SEQ ID NO: 84) MNGNRSIVYREFVGVTPVAKTLRNELRPVGHTQEHIIQNGLIQEDELRQEKSTELKNIMDDYYREYIDKSLSGLTDLDFTLLFELMNSVQSSLSKDNKKALEKEHNKMREQICTHLQSDSDYKNMFNAKLFKEILPDFIKNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVSTSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNNYNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDEHISLIESEEKADEIKKRLDMYMNMYHWVKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYTSKKIKLNFQSPTLANGWSQSKEFDNNAIIIIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLIDYFKNSIEKHAEWRKYEFKFSATDSYNDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFSEENLKNIVIKLNGQAELFYRKASVKNPVKHKKDSVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRTAQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMAVKYIAQNDDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLMVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKGKSVDEPGGLLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYADGIADVRIDMEKMYEDKNSEFFAQLLSLYKLTVQMRNSYTEAEEQEKGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYE>WP_012739647_(modified) hypothetical protein [[Eubacterium] eligens](SEQ ID NO: 85) MNGNRSIVYREFVGVIPVAKTLRNELRPVGHTQEHIIQNGLIQEDELRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSPSKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLLKEILPDFIKNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVSTSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNNYNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDDHISLIESEEKADEMKKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLIDYFKNSIEKHAEWRKYEFKFSATDSYSDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFSEENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRTAQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMVVKYIAQNDDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLIVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKEKSVDEPGGLLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYADGEEDIRIDMEKMDEDKKSEFFAQLLSLYKLTVQMRNSYTEAEEQENGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYE>WP_045971446_(modified) hypothetical protein [Flavobacterium sp. 316](SEQ ID NO: 86) MKNFSNLYQVSKTVRFELKPIGNTLENIKNKSLLKNDSIRAESYQKMKKTIDEFHKYHDLALNNKKLSYLNEYIALYTQSAEAKKEDKFKADFKKVQDNLRKEIVSSFTEGEAKAIFSVLDKKELITIELEKWKNENNLAVYLDESFKSFTTYFTGFHQNRKNMYSAEANSTAIAYRLIHENLPKFIENSKAFEKSSQIAELQPKIEKLYKEFEAYLNVNSISELFEIDYFNEVLTQKGITVYNNIIGGRTATEGKQKIQGLNEIINLYNQTKPKNERLPKLKQLYKQILSDRISLSFLPDAFTEGKQVLKAVFEFYKINLLSYKQDGVEESQNLLELIQQVVKNLGNQDVNKIYLKNDTSLTTIAQQLFGDFSVFSAALQYRYETVVNPKYTAEYQKANEAKQEKLDKEKIKFVKQDYFSIAFLQEVVADYVKTLDENLDWKQKYTPSCIADYFTTHFIAKKENEADKTFNFIANIKAKYQCIQGILEQADDYEDELKQDQKLIDNIKFFLDAILEVVHFIKPLHLKSESITEKDNAFYDVFENYYEALNVVTPLYNMVRNYVTQKPYSTEKIKLNFENAQLLNGWDANKEKDYLTTILKRDGNYFLAIMDKKHNKTFQQFTEDDENYEKIVYKLLPGVNKMLPKVFFSNKNIAFFNPSKEILDNYKNNTEIKKGATFNLKDCHALIDFFKDSLNKHEDWKYFDFQFSETKTYQDLSGFYKEVEHQGYKINFKKVSVSQIDTLIEEGKMYLFQIYNKDFSPYAKGKPNMHTLYWKALFETQNLENVIYKLNGQAEIFFRKASIKKKNIITHKAHQPIAAKNPLTPTAKNTFAYDLIKDKRYTVDKFQFHVPITMNFKATGNSYINQDVLAYLKDNPEVNIIGLDRGERHLVYLTIMQKGTILLQESLNVIQDEKTHTPYHTLLDNKEIARDKARKNWGSIESIKELKEGYISQVVHKITKMMIEHNAIVVMEDLNFGFKRGRFKVEKQIYQKLEKMLIDKLNYLVLKDKQPHELGGLYNALQLTNKFESFQKMGKQSGFLFYVPAWNTSKIDPTTGFVNYFYTKYENVEKAKTFFSKFDSILYNKTKGYFEFVVKNYSDFNPKAADTRQEWTICTHGERIETKRQKEQNNNFVSTTIQLTEQFVNFFEKVGLDLSKELKTQLIAQNEKSFFEELFHLLKLTLQMRNSESHTEIDYLISPVANEKGIFYDSRKATASLPIDADANGAYHIAKKGLWIMEQINKTNSEDDLKKVKLAISNREWLQYVQQVQKK>WP_044110123_(modified) hypothetical protein [Prevotella brevis](SEQ ID NO: 87) MKQFTNLYQLSKTLRFELKPIGKTLEHINANGFIDNDAHRAESYKKVKKLIDDYHKDYIENVLNNFKLNGEYLQAYFDLYSQDTKDKQFKDIQDKLRKSIASALKGDDRYKTIDKKELIRQDMKTFLKKDTDKALLDEFYEFTTYFTGYHENRKNMYSDEAKSTAIAYRLIHDNLPKFIDNIAVFKKIANTSVADNFSTIYKNFEEYLNVNSIDEIFSLDYYNIVLTQTQIEVYNSIIGGRTLEDDTKIQGINEFVNLYNQQLANKKDRLPKLKPLFKQILSDRVQLSWLQEEFNTGADVLNAVKEYCTSYFDNVEESVKVLLTGISDYDLSKIYITNDLALTDVSQRMFGEWSIIPNAIEQRLRSDNPKKTNEKEEKYSDRISKLKKLPKSYSLGYINECISELNGIDIADYYATLGAINTESKQEPSIPTSIQVHYNALKPILDTDYPREKNLSQDKLTVMQLKDLLDDFKALQHFIKPLLGNGDEAEKDEKFYGELMQLWEVIDSITPLYNKVRNYCTRKPFSTEKIKVNFENAQLLDGWDENKESTNASIILRKNGMYYLGIMKKEYRNILTKPMPSDGDCYDKVVYKFFKDITTMVPKCTTQMKSVKEHFSNSNDDYTLFEKDKFIAPVVITKEIFDLNNVLYNGVKKFQIGYLNNTGDSFGYNHAVEIWKSFCLKFLKAYKSTSIYDFSSIEKNIGCYNDLNSFYGAVNLLLYNLTYRKVSVDYIHQLVDEDKMYLFMIYNKDFSTYSKGTPNMHTLYWKMLFDESNLNDVVYKLNGQAEVFYRKKSITYQHPTHPANKPIDNKNVNNPKKQSNFEYDLIKDKRYTVDKFMFHVPITLNFKGMGNGDINMQVREYIKTTDDLHFIGIDRGERHLLYICVINGKGEIVEQYSLNEIVNNYKGTEYKTDYHTLLSERDKKRKEERSSWQTIEGIKELKSGYLSQVIHKITQLMIKYNAIVLLEDLNMGFKRGRQKVESSVYQQFEKALIDKLNYLVDKNKDANEIGGLLHAYQLTNDPKLPNKNSKQSGFLFYVPAWNTSKIDPVTGFVNLLDTRYENVAKAQAFFKKFDSIRYNKEYDRFEFKFDYSNFTAKAEDTRTQWTLCTYGTRIETFRNAEKNSNWDSREIDLTTEWKTLFTQHNIPLNANLKEAILLQANKNFYTDILHLMKLTLQMRNSVTGTDIDYMVSPVANECGEFFDSRKVKEGLPVNADANGAYNIARKGLWLAQQIKNANDLSDVKLAITNKEWLQFAQKKQYLKD>WP_036388671_(modified) hypothetical protein [Moraxella caprae](SEQ ID NO: 88) MLFQDFTHLYPLSKTMRFELKPIGKTLEHIHAKNFLSQDETMADMYQKVKAILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDDGLQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDGKELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYSDEDKHTAITYRLIRENLPRFIDNLQILATIKQKHSALYDQIINELTASGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSRKIQGINELINSHHNQHCHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEMCQAVNEFYRHYADVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDNQDGNFYGEFGALYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGIILQKDGCYYLALLDKAHKKVFDNAPNTGKNVYQKMIYKLLPGPNKMLPKVFFAKSNLDYYNPSAELLDKYAQGTHKKGNNFNLKDCHALIDFFKAGINKHPEWQHFGFKFSPTSSYQDLSDFYREVEPQGYQVKFVDINADYINELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSKDNLANPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQMTTPYIAKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQISQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDEADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADKDYFEFHIDYAKFTDKAKNSRQIWKICSHGDKRYVYDKTANQNKGATKGINVNDELKSLFARHHINDKQPNLVMDICQNNDKEFHKSLIYLLKTLLALRYSNASSDEDFILSPVANDEGMFFNSALADDTQPQNADANGAYHIALKGLWVLEQIKNSDDLNKVKLAIDNQTWLNFAQNR >WP_020988726_(modified) CRISPR-associated protein Cpf1, subtype PREFRAN[Leptospira inadai] (SEQ ID NO: 89)MEDYSGFVNIYSIQKTLRFELKPVGKTLEHIEKKGFLKKDKIRAEDYKAVKKIIDKYHRAYIEEVFDSVLHQKKKKDKTRFSTQFIKEIKEFSELYYKTEKNIPDKERLEALSEKLRKMLVGAFKGEFSEEVAEKYKNLFSKELIRNEIEKFCETDEERKQVSNFKSFTTYFTGFHSNRQNIYSDEKKSTAIGYRIIHQNLPKFLDNLKIIESIQRRFKDFPWSDLKKNLKKIDKNIKLTEYFSIDGFVNVLNQKGIDAYNTILGGKSEESGEKIQGLNEYINLYRQKNNIDRKNLPNVKILFKQILGDRETKSFIPEAFPDDQSVLNSITEFAKYLKLDKKKKSIIAELKKFLSSFNRYELDGIYLANDNSLASISTFLFDDWSFIKKSVSFKYDESVGDPKKKIKSPLKYEKEKEKWLKQKYYTISFLNDAIESYSKSQDEKRVKIRLEAYFAEFKSKDDAKKQFDLLERIEEAYAIVEPLLGAEYPRDRNLKADKKEVGKIKDFLDSIKSLQFFLKPLLSAEIFDEKDLGFYNQLEGYYEEIDSIGHLYNKVRNYLTGKIYSKEKFKLNFENSTLLKGWDENREVANLCVIFREDQKYYLGVMDKENNTILSDIPKVKPNELFYEKMVYKLIPTPHMQLPRIIFSSDNLSIYNPSKSILKIREAKSFKEGKNFKLKDCHKFIDFYKESISKNEDWSRFDFKFSKTSSYENISEFYREVERQGYNLDFKKVSKFYIDSLVEDGKLYLFQIYNKDFSIFSKGKPNLHTIYFRSLFSKENLKDVCLKLNGEAEMFFRKKSINYDEKKKREGHHPELFEKLKYPILKDKRYSEDKFQFHLPISLNFKSKERLNFNLKVNEFLKRNKDINIIGIDRGERNLLYLVMINQKGEILKQTLLDSMQSGKGRPEINYKEKLQEKEIERDKARKSWGTVENIKELKEGYLSIVIHQISKLMVENNAIVVLEDLNIGFKRGRQKVERQVYQKFEKMLIDKLNFLVFKENKPTEPGGVLKAYQLTDEFQSFEKLSKQTGFLFYVPSWNTSKIDPRTGFIDFLHPAYENIEKAKQWINKFDSIRFNSKMDWFEFTADTRKFSENLMLGKNRVWVICTTNVERYFTSKTANSSIQYNSIQITEKLKELFVDIPFSNGQDLKPEILRKNDAVFFKSLLFYIKTTLSLRQNNGKKGEEEKDFILSPVVDSKGRFFNSLEASDDEPKDADANGAYHIALKGLMNLLVLNETKEENLSRPKWKIKNKDWLEFVWER NR>WP_023936172_(modified) exonuclease SbcC [Porphyromonas creyioricanis](SEQ ID NO: 90) MPWIDLKDFTNLYPVSKTLRFELKPVGKTLENIEKAGILKEDEHRAESYRRVKKIIDTYHKVFIDSSLENMAKMGIENEIKAMLQSFCELYKKDHRTEGEDKALDKIRAVLRGLIVGAFTGVCGRRENTVQNEKYESLFKEKLIKEILPDFVLSTEAESLPFSVEEATRSLKEFDSFTSYFAGFYENRKNIYSTKPQSTAIAYRLIHENLPKFIDNILVFQKIKEPIAKELEHIRADFSAGGYIKKDERLEDIFSLNYYIHVLSQAGIEKYNALIGKIVTEGDGEMKGLNEHINLYNQQRGREDRLPLFRPLYKQILSDREQLSYLPESFEKDEELLRALKEFYDHIAEDILGRTQQLMTSISEYDLSRIYVRNDSQLTDISKKMLGDWNAIYMARERAYDHEQAPKRITAKYERDRIKALKGEESISLANLNSCIAFLDNVRDCRVDTYLSTLGQKEGPHGLSNLVENVFASYHEAEQLLSFPYPEENNLIQDKDNVVLIKNLLDNISDLQRFLKPLWGMGDEPDKDERFYGEYNYIRGALDQVIPLYNKVRNYLTRKPYSTRKVKLNFGNSQLLSGWDRNKEKDNSCVILRKGQNFYLAIMNNRHKRSFENKVLPEYKEGEPYFEKMDYKFLPDPNKMLPKVFLSKKGIEIYEPSPKLLEQYGHGTIAKKGDTFSMDDLHELIDFFKHSIEAHEDWKQFGFKFSDTATYENVSSFYREVEDQGYKLSFRKVSESYVYSLIDQGKLYLFQIYNKDFSPCSKGTPNLHTLYWRMLFDERNLADVIYKLDGKAEIFFREKSLKNDHPTHPAGKPIKKKSRQKKGEESLFEYDLVKDRRYTMDKFQFHVPITMNFKCSAGSKVNDMVNAHIREAKDMHVIGIDRGERNLLYICVIDSRGTILDQISLNTINDIDYHDLLESRDKDRQQERRNWQTIEGIKELKQGYLSQAVHRIAELMVAYKAVVALEDLNMGFKRGRQKVESSVYQQFEKQLIDKLNYLVDKKKRPEDIGGLLRAYQFTAPFKSFKEMGKQNGFLFYIPAWNTSNIDPTTGFVNLFHAQYENVDKAKSFFQKFDSISYNPKKDWFEFAFDYKNFTKKAEGSRSMWILCTHGSRIKNFRNSQKNGQWDSEEFALTEAFKSLFVRYEIDYTADLKTAIVDEKQKDFFVDLLKLFKLTVQMRNSWKEKDLDYLISPVAGADGRFFDTREGNKSLPKDADANGAYNIALKGLWALRQIRQTSEGGKLKLAISNKEWLQFVQERSYEKD>WP_009217842_(modified) hypothetical protein [Bacteroidetes oral taxon 274](SEQ ID NO: 91) MRKFNEFVGLYPISKTLRFELKPIGKTLEHIQRNKLLEHDAVRADDYVKVKKIIDKYHKCHDEALSGFTFDTEADGRSNNSLSEYYLYYNLKKRNEQEQKTFKTIQNNLRKQIVNKLTQSEKYKRIDKKELITTDLPDFLTNESEKELVEKFKNFTTYFTEFHKNRKNMYSKEEKSTAIAFRLINENLPKFVDNIAAFEKVVSSPLAEKINALYEDFKEYLNVEEISRVFRLDYYDELLTQKQIDLYNAIVGGRTEEDNKIQIKGLNQYINEYNQQQTDRSNRLPKLKPLYKQILSDRESVSWLPPKFDSDKNLLIKIKECYDALSEKEKVFDKLESILKSLSTYDLSKIYISNDSQLSYISQKMFGRWDIISKAIREDCAKRNPQKSRESLEKFAERIDKKLKTIDSISIGDVDECLAQLGETYVKRVEDYFVAMGESEIDDEQTDTTSFKKNIEGAYESVKELLNNADNITDNNLMQDKGNVEKIKTLLDAIKDLQRFIKPLLGKGDEADKDGVFYGEFTSLWTKLDQVTPLYNMVRNYLTSKPYSTKKIKLNFENSTLMDGWDLNKEPDNTTVIFCKDGLYYLGIMGKKYNRVFVDREDLPHDGECYDKMEYKLLPGANKMLPKVFFSETGIQRFLPSEELLGKYERGTHKKGAGFDLGDCRALIDFFKKSIERHDDWKKFDFKFSDTSTYQDISEFYREVEQQGYKMSFRKVSVDYIKSLVEEGKLYLFQIYNKDFSAHSKGTPNMHTLYWKMLFDEENLKDVVYKLNGEAEVFFRKSSITVQSPTHPANSPIKNKNKDNQKKESKFEYDLIKDRRYTVDKFLFHVPITMNFKSVGGSNINQLVKRHIRSATDLHIIGIDRGERHLLYLTVIDSRGNIKEQFSLNEIVNEYNGNTYRTDYHELLDTREGERTEARRNWQTIQNIRELKEGYLSQVIHKISELAIKYNAVIVLEDLNFGFMRSRQKVEKQVYQKFEKMLIDKLNYLVDKKKPVAETGGLLRAYQLTGEFESFKTLGKQSGILFYVPAWNTSKIDPVTGFVNLFDTHYENIEKAKVFFDKFKS1RYNSDKDWFEFVVDDYTRFSPKAEGTRRDWTICTQGKRIQICRNHQRNNEWEGQEIDLTKAFKEHFEAYGVDISKDLREQINTQNKKEFFEELLRLLRLTLQMRNSMPSSDIDYLISPVANDTGCFFDSRKQAELKENAVLPMNADANGAYNIARKGLLAIRKMKQEENDSAKISLAISNKEWLKFAQTKPYLED>WP_036890108_(modified) hypothetical protein [Porphyromonas creyioricanis](SEQ ID NO: 92) MDSLKDFTNLYPVSKTLRFELKPVGKTLENIEKAGILKEDEHRAESYRRVKKIIDTYHKVFIDSSLENMAKMGIENEIKAMLQSFCELYKKDHRTEGEDKALDKIRAVLRGLIVGAFTGVCGRRENTVQNEKYESLFKEKLIKElLPDFVLSTEAESLPFSVEEATRSLKEFDSFTSYFAGFYENRKNIYSTKPQSTAIAYRLIHENLPKFIDNILVFQKIKEPIAKELEHIRADFSAGGYIKKDERLEDIFSLNYYIHVLSQAGIEKYNALIGKIVTEGDGEMKGLNEHINLYNQQRGREDRLPLFRPLYKQILSDREQLSYLPESFEKDEELLRALKEFYDHIAEDILGRTQQLMTSISEYDLSRIYVRNDSQLTDISKKMLGDWNAIYMARERAYDHEQAPKRITAKYERDRIKALKGEESISLANLNSCIAFLDNVRDCRVDTYLSTLGQKEGPHGLSNLVENVFASYHEAEQLLSFPYPEENNLIQDKDNVVLIKNLLDNISDLQRFLKPLWGMGDEPDKDERFYGEYNYIRGALDQVIPLYNKVRNYLTRKPYSTRKVKLNFGNSQLLSGWDRNKEKDNSCVILRKGQNFYLAIMNNRHKRSFENKMLPEYKEGEPYFEKMDYKFLPDPNKMLPKVFLSKKGIEIYKPSPKLLEQYGHGTHKKGDTFSMDDLHELIDFFKHSIEAHEDWKQFGFKFSDTATYENVSSFYREVEDQGYKLSFRKVSESYVYSLIDQGKLYLFQIYNKDFSPCSKGTPNLHTLYWRMLFDERNLADVIYKLDGKAEIFFREKSLKNDHPTHPAGKPIKKKSRQKKGEESLFEYDLVKDRRYTMDKFQFHVPITMNFKCSAGSKVNDMVNAHIREAKDMHVIGIDRGERNLLYICVIDSRGTILDQISLNTINDIDYHDLLESRDKDRQQEHRNWQTIEGIKELKQGYLSQAVHRIAELMVAYKAVVALEDLNMGFKRGRQKVESSVYQQFEKQLIDKLNYLVDKKKRPEDIGGLLRAYQFTAPFKSFKEMGKQNGFLFYIPAWNTSNIDPTTGFVNLFHVQYENVDKAKSFFQKFDSISYNPKKDWFEFAFDYKNFTKKAEGSRSMWILCTHGSRIKNFRNSQKNGQWDSEEFALTEAFKSLFVRYEIDYTADLKTAIVDEKQKDFFVDLLKLFKLTVQMRNSWKEKDLDYLISPVAGADGRFFDTREGNKSLPKDADANGAYNIALKGLWALRQIRQTSEGGKLKLAISNKEWLQFVQERSYEKD>WP_036887416_(modified) hypothetical protein [Porphyromonas crevioricanis](SEQ ID NO: 93) MDSLKDFTNLYPVSKTLRFELKPVGKTLENIEKAGILKEDEHRAESYRRVKKIIDTYHKVFIDSSLENMAKMGIENEIKAMLQSFCELYKKDHRTEGEDKALDKIRAVLRGLIVGAFTGVCGRRENTVQNEKYESLFKEKLIKEILPDFVLSTEAESLPFSVEEATRSLKEFDSFTSYFAGFYENRKNIYSTKPQSTAIAYRLIHENLPKFIDNILVFQKIKEPIAKELEHIRADFSAGGYIKKDERLEDIFSLNYYIHVLSQAGIEKYNALIGKIVTEGDGEMKGLNEHINLYNQQRGREDRLPLFRPLYKQILSDREQLSYLPESFEKDEELLRALKEFYDHIAEDILGRTQQLMTSISEYDLSRIYVRNDSQLTDISKKMLGDWNAIYMARERAYDHEQAPKRITAKYERDRIKALKGEESISLANLNSCIAFLDNVRDCRVDTYLSTLGQKEGPHGLSNLVENVFASYHEAEQLLSFPYPEENNLIQDKDNVVLIKNLLDNISDLQRFLKPLWGMGDEPDKDERFYGEYNYIRGALDQVIPLYNKVRNYLTRKPYSTRKVKLNFGNSQLLSGWDRNKEKDNSCVILRKGQNFYLAIMNNRHKRSFENKVLPEYKEGEPYFEKMDYKFLPDPNKMLPKVFLSKKGIEIYKPSPKLLEQYGHGTHKKGDTFSMDDLHELIDFFKHSLEAHEDWKQFGFKFSDTATYENVSSFYREVEDQGYKLSFRKVSESYVYSLIDQGKLYLFQIYNKDFSPCSKGTPNLHTLYWRMLFDERNLADVIYKLDGKAEIFFREKSLKNDHPTHPAGKPIKKKSRQKKGEESLFEYDLVKDRHYTMDKFQFHVPITMNFKCSAGSKVNDMVNAHIREAKDMHVIGIDRGERNLLYICVIDSRGTILDQISLNTINDIDYHDLLESRDKDRQQERRNWQTIEGIKELKQGYLSQAVHRIAELMVAYKAVVALEDLNMGFKRGRQKVESSVYQQFEKQLIDKLNYLVDKKKRPEDIGGLLRAYQFTAPFKSFKEMGKQNGFLFYIPAWNTSNIDPTTGFVNLFHAQYENVDKAKSFFQKFDSISYNPKKDWFEFAFDYKNFTKKAEGSRSMWILCTHGSRIKNFRNSQKNGQWDSEEFALTEAFKSLFVRYEIDYTADLKTAIVDEKQKDFFVDLLKLFKLTVQMRNSWKEKDLDYLISPVAGADGRFFDTREGNKSLPKDADANGAYNIALKGLWALRQIRQTSEGGKLKLAISNKEWLQFVQERSYEKD>WP_023941260_(modified) exonuclease SbcC [Porphyromonas cansulci](SEQ ID NO: 94) MDSLKDFTNLYPVSKTLRFELKPVGKTLENIEKAGILKEDEHRAESYRRVKKIIDTYHKVFIDSSLENMAKMGIENEIKAMLQSFCELYKKDHRTEGEDKALDKIRAVLRGLIVGAFTGVCGRRENTVQNEKYESLFKEKLIKEILPDFVLSTEAESLPFSVEEATRSLKEFDSFTSYFAGFYENRKNIYSTKPQSTAIAYRLIHENLPKFIDNILVFQKIKEPIAKELEHIRADFSAGGYIKKDERLEDIFSLNYYIHVLSQAGIEKYNALIGKIVTEGDGEMKGLNEHINLYNQQRGREDRLPLFRPLYKQILSDREQLSYLPESFEKDEELLRALKEFYDHIAEDILGRTQQLMTSISEYDLSRIYVRNDSQLTDISKKMLGDWNAIYMARERAYDHEQAPKRITAKYERDRIKALKGEESISLANLNSCIAFLDNVRDCRVDTYLSTLGQKEGPHGLSNLVENVFASYHEAEQLLSFPYPEENNLIQDKDNVVLIKNLLDNISDLQRFLKPLWGMGDEPDKDERFYGEYNYIRGALDQVIPLYNKVRNYLTRKPYSTRKVKLNFGNSQLLSGWDRNKEKDNSCVILRKGQNFYLAIMNNRHKRSFENKVLPEYKEGEPYFEKMDYKFLPDPNKMLPKVFLSKKGIEIYKPSPKLLEQYGHGTHKKGDTFSMDDLHELIDFFKHSIEAHEDWKQFGFKFSDTATYENVSSFYREVEDQGYKLSFRKVSESYVYSLIDQGKLYLFQIYNKDFSPCSKGTPNLHTLYWRMLFDERNLADVIYKLDGKAEIFFREKSLKNDHPTHPAGKPIKKKSRQKKGEESLFEYDLVKDRRYTMDKFQFHVPITMNFKCSAGSKVNDMVNAHIREAKDMHVIGIDRGERNLLYICVIDSRGTILDQISLNTINDIDYHDLLESRDKDRQQERRNWQTIEGIKELKQGYLSQAVHRIAELMVAYKAVVALEDLNMGFKRGRQKVESSVYQQFEKQLIDKLNYLVDKKKRPEDIGGLLRAYQFTAPFKSFKEMGKQNGFLFYIPAWNTSNIDPTTGFVNLFHAQYENVDKAKSFFQKFDSISYNPKKDWFEFAFDYKNFTKKAEGSRSMWILCTHGSRIKNFRNSQKNGQWDSEEFALTEAFKSLFVRYEIDYTADLKTAIVDEKQKDFFVDLLKLFKLTVQMRNSWKEKDLDYLISPVAGADGRFFDTREGNKSLPKDADANGAYNIALKGLWALRQIRQTSEGGKLKLAISNKEWLQFVQERSYEKD>WP_037975888_(modified) hypothetical protein [Synergistes jonesii](SEQ ID NO: 95) MANSLKDFTNIYQLSKTLRFELKPIGKTEEHINRKLIIMHDEKRGEDYKSVTKLIDDYHRKFIHETLDPAHFDWNPLAEALIQSGSKNNKALPAEQKEMREKIISMFTSQAVYKKLFKKELFSELLPEMIKSELVSDLEKQAQLDAVKSFDKFSTYFTGFHENRKNIYSKKDTSTSIAFRIVHQNFPKFLANVRAYTLIKERAPEVIDKAQKELSGILGGKTLDDIFSIESFNNVLTQDKIDYYNQIIGGVSGKAGDKKLRGVNEFSNLYRQQHPEVASLRIKMVPLYKQILSDRTTLSFVPEALKDDEQAINAVDGLRSELERNDIFNRIKRLFGKNNLYSLDKIWIKNSSISAFSNELFKNWSFIEDALKEFKENEFNGARSAGKKAEKWLKSKYFSFADIDAAVKSYSEQVSADISSAPSASYFAKFTNLIETAAENGRKFSYFAAESKAFRGDDGKTEIIKAYLDSLNDILHCLKPFETEDISDIDTEFYSAFAEIYDSVKDVIPVYNAVRNYTTQKPFSTEKFKLNFENPALAKGWDKNKEQNNTAIILMKDGKYYLGVIDKNNKLRADDLADDGSAYGYMKMNYKFIPTPHMELPKVFLPKRAPKRYNPSREILLIKENKTFIKDKNFNRTDCHKLIDFFKDSINKHKDWRTFGFDFSDTDSYEDISDFYMEVQDQGYKLTFTRLSAEKIDKWVEEGRLFLFQIYNKDFADGAQGSPNLHTLYWKAIFSEENLKDVVLKLNGEAELFFRRKSIDKPAVHAKGSMKVNRRDIDGNPIDEGTYVEICGYANGKRDMASLNAGARGLIESGLVRITEVKHELVKDKRYTIDKYFFHVPFTINFKAQGQGNINSDVNLFLRNNKDVNIIGIDRGERNLVYVSLIDRDGHIKLQKDFNIIGGMDYHAKLNQKEKERDTARKSWKTIGTIKELKEGYLSQVVREIVRLAVDNNAVIVMEDLNIGFKRGRFKVEKQVYQKFEKMLIDKLNYLVFKDAGYDAPCGILKGLQLTEKFESFTKLGKQCGIIFYIPAGYTSKIDPTTGFVNLFNINDVSSKEKQKDFIGKLDSIRFDAKRDMFTFEFDYDKFRTYQTSYRKKWAVWTNGKRIVREKDKDGKFRMNDRLLTEDMKNILNKYALAYKAGEDILPDVISRDKSLASEIFYVFKNTLQMTNSKRDTGEDFIISPVLNAKGRFFDSRKTDAALPIDADANGAYHIALKGSLVLDAIDEKLKEDGRIDYKDMAVSNPKWFEFMQTRKFDF>EFI70750_(modified) conserved hypothetical protein [Prevotella bryantii B14](SEQ ID NO: 96) MQINNLKIIYMKFTDFTGLYSLSKTLRFELKPIGKTLENIKKAGLLEQDQHRADSYKKVKKIIDEYHKAFIEKSLSNFELKYQSEDKLDSLEEYLMYYSMKRIEKTEKDKFAKIQDNLRKQIADHLKGDESYKTIFSKDIIRKNLPDFVKSDEERTHKEFKDFTTYFKGFYENRENMYSAEDKSTAISHRIIHENLPKFVDNINAFSKIILIPELREKLNQIYQDFEEYLNVESIDEIFHLDYFSMVMTQKQIEVYNAIIGGKSTNDKKIQGLNEYINLYNQKHKDCKLPKLKLLFKQILSDRIAISWLPDNFKDDQEALDSIDTCYKNLLNDGNVLGEGNLKLLLENIDTYNLKGIFIRNDLQLTDISQKMYASWNVIQDAVILDLKKQVSRKKKESAEDYNDRLKKLYTSQESFSIQYLNDCLRAYGKTENIQDYFAKLGAVNNEHEQTINLFAQVRNAYTSVQAILTTPYPENANLAQDKETVALIKNLLDSLKRLQRFIKPLLGKGDESDKDERFYGDFTPLWETLNQITPLYNMVRNYMTRKPYSQEKIKLNFENSTLLGGWDLNKEHDNTAIILRKNGLYYLAIMKKSANKIFDKDKLDNSGDCYEKMVYKLLPGANKMLPKVFFSKSRIDEFKPSENIIENYKKGTHKKGANFNLADCHNLIDFFKSSISKHEDWSKFNFHFSDTSSYEDLSDFYREVEQQGYSISFCDVSVEYINKMVEKGDLYLFQIYNKDFSEFSKGTPNMHTLYWNSLFSKENLNNIIYKLNGQAEIFFRKKSLNYKRPTHPAHQAIKNKNKCNEKKESIFDYDLVKDKRYTVDKFQFHVPITMNFKSTGNTNINQQVIDYLRTEDDTHIIGIDRGERHLLYLVVIDSHGKIVEQFTLNEIVNEYGGNIYRTNYHDLLDTREQNREKARESWQTIENIKELKEGYISQVIHKITDLMQKYHAVVVLEDLNMGFMRGRQKVEKQVYQKFEEMLINKLNYLVNKKADQNSAGGLLHAYQLTSKFESFQKLGKQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYESIDKAKAFFGKFDSIRYNADKDWFEFAFDYNNFTTKAEGTRTNWTICTYGSRIRTFRNQAKNSQWDNEEIDLTKAYKAFFAKHGINIYDNIKEAIAMETEKSFFEDLLHLLKLTLQMRNSITGTTTDYLISPVHDSKGNFYDSRICDNSLPANADANGAYNIARKGLMLIQQIKDSTSSNRFKFSPITNKDWLIFA QEKPYLND>WP_024988992_(modified) hypothetical protein [Prevotella albensis](SEQ ID NO: 97) MNIKNFTGLYPLSKTLRFELKPIGKTKENIEKNGILTKDEQRAKDYLIVKGFIDEYHKQFIKDRLWDFKLPLESEGEKNSLEEYQELYELTKRNDAQEADFTEIKDNLRSSITEQLTKSGSAYDRIFKKEFIREDLVNFLEDEKDKNIVKQFEDFTTYFTGFYENRKNMYSSEEKSTAIAYRLIHQNLPKFMDNMRSFAKIANSSVSEHFSDIYESWKEYLNVNSIEEIFQLDYFSETLTQPHIEVYNYIIGKKVLEDGTEIKGINEYVNLYNQQQKDKSKRLPFLVPLYKQILSDREKLSWIAEEFDSDKKMLSAITESYNHLHNVLMGNENESLRNLLLNIKDYNLEKINITNDLSLTEISQNLFGRYDVFTNGIKNKLRVLTPRKKKETDENFEDRINKIFKTQKSFSIAFLNKLPQPEMEDGKPRNIEDYFITQGAINTKSIQKEDIFAQIENAYEDAQVFLQIKDTDNKLSQNKTAVEKIKTLLDALKELQHFIKPLLGSGEENEKDELFYGSFLAIWDELDTITPLYNKVRNWLTRKPYSTEKIKLNFDNAQLLGGWDVNKEHDCAGILLRKNDSYYLGIINKKTNHIFDTDITPSDGECYDKIDYKLLPGANKMLPKVFFSKSRIKEFEPSEAIINCYKKGTHKKGKNFNLTDCHRLINFFKTSIEKHEDWSKFGFKFSDTETYEDISGFYREVEQQGYRLTSHPVSASYIHSLVKEGKLYLFQIWNKDFSQFSKGTPNLHTLYWKMLFDKRNLSDVVYKLNGQAEVFYRKSSIEHQNRIIHPAQHPITNKNELNKKHTSTFKYDIIKDRRYTVDKFQFHVPITINFKATGQNNINPIVQEVIRQNGITHIIGIDRGERHLLYLSLIDLKGNIIKQMTLNEIINEYKGVTYKTNYHNLLEKREKERTEARHSWSSIESIKELKDGYMSQVIHKITDMMVKYNAIVVLEDLNGGFMRGRQKVEKQVYQKFEKKLIDKLNYLVDKKLDANEVGGVLNAYQLTNKFESFKKIGKQSGFLFYIPAWNTSKIDPITGFVNLFNTRYESIKETKVFWSKFDIIRYNKEKNWFEFVFDYNTFTTKAEGTRTKWTLCTHGTRIQTFRNPEKNAQWDNKEINLTESFKALFEKYKIDITSNLKESIMQETEKKFFQELHNLLHLTLQMRNSVTGTDIDYLISPVADEDGNFYDSRINGKNFPENADANGAYNIARKGLMLIRQIKQADPQKKFKFETITNKDWLKFAQDKPYLKD>WP_039658684_(modified) hypothetical protein [Smithella sp. SC_K08D17](SEQ ID NO: 98) MQTLFENFTNQYPVSKTLRFELIPQGKTKDFIEQKGLLKKDEDRAEKYKKVKNIIDEYHKDFIEKSLNGLKLDGLEKYKTLYLKQEKDDKDKKAFDKEKENLRKQIANAFRNNEKFKTLFAKELIKNDLMSFACEEDKKNVKEFEAFTTYFTGFHQNRANMYVADEKRTAIASRLIHENLPKFIDNIKIFEKMKKEAPELLSPFNQTLKDMKDVIKGTTLEEIFSLDYFNKTLTQSGIDIYNSVIGGRTPEEGKTKIKGLNEYINTDFNQKQTDKKKRQPKFKQLYKQILSDRQSLSFIAEAFKNDTEILEAIEKFYVNELLHFSNEGKSTNVLDAIKNAVSNLESFNLTKMYFRSGASLTDVSRKVFGEWSIINRALDNYYATTYPIKPREKSEKYEERKEKWLKQDFNVSLIQTAIDEYDNETVKGKNSGKVIADYFAKFCDDKETDLIQKVNEGYIAVKDLLNTPCPENEKLGSNKDQVKQIKAFMDSIMDIMHFVRPLSLKDTDKEKDETFYSLFTPLYDHLTQTIALYNKVRNYLTQKPYSTEKIKLNFENSTLLGGWDLNKETDNTAIILRKDNLYYLGIMDKRHNRIFRNVPKADKKDFCYEKMVYKLLPGANKMLPKVFFSQSRIQEFTPSAKLLENYANETHKKGDNFNLNHCHKLIDFFKDSINKHEDWKNFDFRFSATSTYADLSGFYHEVEHQGYKISFQSVADSFIDDLVNEGKLYLFQIYNKDFSPFSKGKPNLHTLYWKMLFDENNLKDVVYKLNGEAEVFYRKKSIAEKNTTIHKANESIINKNPDNPKATSTFNYDIVKDKRYTIDKFQFHIPITMNFKAEGIFNMNQRVNQFLKANPDINIIGIDRGERHLLYYALINQKGKILKQDTLNVIANEKQKVDYHNLLDKKEGDRATARQEWGVIETIKELKEGYLSQVIHKLTDLMIENNAIIVMEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVDKNKKANELGGLLNAFQLANKFESFQKMGKQNGFIFYVPAWNTSKTDPATGFIDFLKPRYENLNQAKDFFEKFDSIRLNSKADYFEFAFDFKNFTEKADGGRTKWTVCTTNEDRYAWNRALNNNRGSQEKYDITAELKSLFDGKVDYKSGKDLKQQIASQESADFFKALMKNLSITLSLRHNNGEKGDNEQDYILSPVADSKGRFFDSRKADDDMPKNADANGAYHIALKGLWCLEQISKTDDLKKVKLAISNKE WLEFVQTLKG>WP_037385181_(modified) hypothetical protein [Smithella sp. SCADC](SEQ ID NO: 99) MQTLFENFTNQYPVSKTLRFELIPQGKTKDFIEQKGLLKKDEDRAEKYKKVKNIIDEYIAKDFIEKSLNGLKLDGLEEYKTLYLKQEKDDKDKKAFDKEKENLRKQIANAFRNNEKFKTLFAKELIKNDLMSFACEEDKKNVKEFEAFTTYFTGFHQNRANMYVADEKRTAIASRLIHENLPKFIDNIKIFEKMKKEAPELLSPFNQTLKDMKDVIKGTTLEEIFSLDYFNKTLTQSGIDIYNSVIGGRTPEEGKTKIKGLNEYINTDFNQKQTDKKKRQPKFKQLYKQILSDRQSLSFIAEAFKNDTEILEAIEKFYVNELLHFSNEGKSTNVLDAIKNAVSNLESFNLTKIYFRSGTSLTDVSRKVFGEWSIINRALDNYYATTYPIKPREKSEKYEERKEKWLKQDFNVSLIQTAIDEYDNETVKGKNSGKVIVDYFAKFCDDKETDLIQKVNEGYIAVKDLLNTPYPENEKLGSNKDQVKQIKAFMDSIMDIMHFVRPLSLKDTDKEKDETFYSLFTPLYDHLTQTIALYNKVRNYLTQKPYSTEKIKLNFENSTLLGGWDLNKETDNTAIILRKENLYYLGIMDKRHNRIFRNVPKADKKDSCYEKMVYKLLPGANKMLPKVFFSQSRIQEFTPSAKLLENYENETHKKGDNFNLNHCHQLIDFFKDSINKHEDWKNFDFRFSATSTYADLSGFYHEVEHQGYKISFQSIADSFIDDLVNEGKLYLFQIYNKDFSPFSKGKPNLHTLYWKMLFDENNLKDVVYKLNGEAEVFYRKKSIAEKNTTIHKANESIINKNPDNPKATSTFNYDIVKDKRYTIDKFQFHVPITMNFKAEGIFNMNQRVNQFLKANPDINIIGIDRGERHLLYYTLINQKGKILKQDTLNVIANEKQKVDYHNLLDKKEGDRATARQEWGVIETIKELKEGYLSQVIHKLTDLMIENNAIIVMEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVDKNKKANELGGLLNAFQLANKFESFQKMGKQNGFIFYVPAWNTSKTDPATGFIDFLKPRYENLKQAKDFFEKFDSIRLNSKADYFEFAFDFKNFTGKADGGRTKWTVCTTNEDRYAWNRALNNNRGSQEKYDITAELKSLFDGKVDYKSGKDLKQQIASQELADFFRTLMKYLSVTLSLRHNNGEKGETEQDYILSPVADSMGKFFDSRKAGDDMPKNADANGAYHIALKGLWCLEQISKTDDLKKVKLAISNK EWLEFMQTLKG>WP_039871282_(modified) hypothetical protein [Prevotella bryantii](SEQ ID NO: 100) MKFTDFTGLYSLSKTLRFELKPIGKTLENIKKAGLLEQDHRADSYKKVKKIIDEYHKAFIEKSLSNFELKYQSEDKLDSLEEYLMYYSMKRIEKTEKDKFAKIQDNLRKQIADHLKGDESYKTIFSKDLIRKNLPDFVKSDEERTLIKEFKDFTTYFKGFYENRENMYSAEDKSTAISHRIIHENLPKFVDNINAFSKIILIPELREKLNQIYQDFEEYLNVESIDEIFHLDYFSMVMTQKQIEVYNAIIGGKSTNDKKIQGLNEYINLYNQKHKDCKLPKLKLLFKQILSDRIAISWLPDNFKDDQEALDSIDTCYKNLLNDGNVLGEGNLKLLLENIDTYNLKGIFIRNDLQLTDISQKMYASWNVIQDAVILDLKKQVSRKKKESAEDYNDRLKKLYTSQESFSIQYLNDCLRAYGKTENIQDYFAKLGAVNNEHEQTINLFAQVRNAYTSVQAILTTPYPENANLAQDKETVALIKNLLDSLKRLQRFIKPLLGKGDESDKDERFYGDFTPLWETLNQITPLYNMVRNYMTRKPYSQEKIKLNFENSTLLGGWDLNKEHDNTAIILRKNGLYYLAIMKKSANKIFDKDKLDNSGDCYEKMVYKLLPGANKMLPKVFFSKSRIDEFKPSENIIENYKKGTHKKGANFNLADCHNLIDFFKSSISKHEDWSKFNFHFSDTSSYEDLSDFYREVEQQGYSISFCDVSVEYINKMVEKGDLYLFQIYNKDFSEFSKGTPNMHTLYWNSLFSKENLNNIIYKLNGQAEIFFRKKSLNYKRPTHPAHQAIKNKNKCNEKKESIFDYDLVKDKRYTVDKFQFHVPITMNFKSTGNTNINQQVIDYLRTEDDTHIIGIDRGERHLLYLVVIDSHGKIVEQFTLNEIVNEYGGNIYRTNYHDLLDTREQNREKARESWQTIENIKELKEGYISQVIHKITDLMQKYHAVVVLEDLNMGFMRGRQKVEKQVYQKFEEMLINKLNYLVNKKADQNSAGGLLHAYQLTSKFESFQKLGKQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYESIDKAKAFFGKFDSIRYNADKDWFEFAFDYNNFTTKAEGTRTNWTICTYGSRIRTFRNQAKNSQWDNEEIDLTKAYKAFFAKHGINIYDNIKEAIAMETEKSFFEDLLHLLKLTLQMRNSITGTTTDYLISPVHDSKGNFYDSRICDNSLPANADANGAYNIARKGLMLIQQIKDSTSSNRFKFSPITNKDWLIFAQEKPYLND >EKE28449_(modified) hypothetical protein ACD_3C00058G0015 [unculturedbacterium (geode 4)] (SEQ ID NO: 101)MFKGDAFTGLYEVQKTLRFELVPIGLTQSYLENDWVIQKDKEVEENYGKIKAYFDLIHKEFVRQSLENAWLCQLDDFYEKYIELHNSLETRKDKNLAKQFEKVMKSLKKEFVSFFDAKWNEWKQKFSFLKKWWIDVLNEKEVLDLMAEFYPDEKELFDKFDKFFTYFSNFKESRKNFYADDGRAWAIATRAIDENLITFIKNIEDFKKLNSSFREFVNDNFSEEDKQIFEIDFYNNCLLQPWIDKYNKIVWWYSLENWEKVQWLNEKINNFKQNQNKSNSKDLKFPRMKLLYKQILGDKEKKVYIDEIRDDKNLIDLIDNSKRRNQIKIDNANDIINDFINNNAKFELDKIYLTRQSINTISSKYFSSWDYIRWYFWTGELQEFVSFYDLKETFWKIEYETLENIFKDCYVKGINTESQNNIVFETQGIYENFLNIFKFEFNQNISQISLLEWELDKIQNEDIKKNEKQVEVIKNYFDSVMSVYKMTKYFSLEKWKKRVELDTDNNFYNDFNEYLEGFEIWKDYNLVRNYITKKQVNTDKIKLNFDNSQFLTWWDKDKENERLGIILRREWKYYLW1LKKWNTLNFGDYLQKEWEIFYEKMNYKQLNNVYRQLPRLLFPLTKKLNELKWDELKKYLSKYIQNFWYNEEIAQIKIEFDIFQESKEKWEKFDIDKLRKLIEYYKKWVLALYSDLYDLEFIKYKNYDDLSIFYSDVEKKMYNLNFTKIDKSLIDGKVKSWELYLFQIYNKDFSESKKEWSTENIHTKYFKLLFNEKNLQNLVVKLSWWADIFFRDKTENLKFKKDKNGQEILDHRRFSQDKIMFHISITLNANCWDKYWFNQYVNEYMNKERDIKIIWIDRWEKHLAYYCVIDKSWKIFNNEIWTLNELNWVNYLEKLEKIESSRKDSRISWWEIENIKELKNGYISQVINKLTELIVKYNAIIVFEDLNIWFKRWRQKIEKQIYQKLELALAKKLNYLTQKDKKDDEILWNLKALQLVPKVNDYQDIWNYKQSWIMFYVRANYTSVTCPNCWLRKNLYISNSATKENQKKSLNSIAIKYNDWKFSFSYEIDDKSWKQKQSLNKKKFIVYSDIERFVYSPLEKLTKVIDVNKKLLELFRDFNLSLDINKQIQEKDLDSVFFKSLTHLFNLILQLRNSDSKDNKDYISCPSCYYHSNNWLQWFEFNWDANWAYNIARKGIILLDRIRKNQEKPDLYVSDIDWDNFVQSNQFPNTIIPIQNIEKQ VPLNIKI>WP_018359861_(modified) hypothetical protein [Porphyromonas macacae](SEQ ID NO: 102) MKTQHFFEDFTSLYSLSKTIRFELKPIGKTLENIKKNGLIRRDEQRLDDYEKLKKVIDEYHEDFIANILSSFSFSEEILQSYIQNLSESEARAKIEKTMRDTLAKAFSEDERYKSIFKKELVKKDIPVWCPAYKSLCKKFDNFTTSLVPFEEENRKNLYTSNEITASIPYRIVHVNLPKFIQNIEALCELQKKMGADLYLEMMENLRNVWPSFVKTPDDLCNLKTYNHLMVQSSISEYNRFVGGYSTEDGTKHQGINEWINIYRQRNKEMRLPGLVFLHKQILAKVDSSSFISDTLENDDQVFCVLRQFRKLFWNTVSSKEDDAASLKDLFCGLSGYDPEAIYVSDAHLATISKNIFDRWNYISDAIRRKTEVLMPRKKESVERYAEKISKQIKKRQSYSLAELDDLLAHYSEESLPAGFSLLSYFTSLGGQKYLVSDGEVILYEEGSNIWDEVLIAFRDLQVILDKDFTEKKLGKDEEAVSVIKKALDSALRLRKFFDLLSGTGAEIRRDSSFYALYTDRMDKLKGLLKMYDKVRNYLTKKPYSIEKFKLHFDNPSLLSGWDKNKELNNLSVIFRQNGYYYLGIMTPKGKNLFKTLPKLGAEEMFYEKMEYKQIAEPMLMLPKVFFPKKTKPAFAPDQSVVDIYNKKTFKTGQKGFNKKDLYRLIDFYKEALTVHEWKLFNFSFSPTEQYRNIGEFFDEVREQAYKVSMVNVPASYIDEAVENGKLYLFQIYNKDFSPYSKGIPNLHTLYWKALFSEQNQSRVYKLCGGGELFYRKASLHMQDTTHPKGISIHKKNLNKKGETSLFNYDLVKDKRFTEDKFFFHVPISINYKNKKITNVNQMVRDYIAQNDDLQIIGIDRGERNLLYISRIDTRGNLLEQFSLNVIESDKGDLRTDYQKILGDREQERLRRRQEWKSIESIKDLKDGYMSQVVHKICNMVVEHKAIVVLENLNLSFMKGRKKVEKSVYEKFERMLVDKLNYLVVDKKNLSNEPGGLYAAYQLTNPLFSFEELHRYPQSGILFFVDPWNTSLTDPSTGFVNLLGRINYTNVGDARKFFDRFNAIRYDGKGNILFDLDLSRFDVRVETQRKLWTLTTFGSRIAKSKKSGKWMVERIENLSLCFLELFEQFNIGYRVEKDLKKAILSQDRKEFYVRLIYLFNLMMQIRNSDGEEDYILSPALNEKNLQFDSRLIEAKDLPVDADANGAYNVARKGLMVVQRIKRGDRESIHRIGRAQWLRYVQE GIVE>WP_013282991_(modified) hypothetical protein [Butyrivibrio proteoclasticus](SEQ ID NO: 103) MLLYENYTKRNQITKSLRLELRPQGKTLRNIKELNLLEQDKAIYALLERLKPVIDEGIKDIARDTLKNCELSFEKLYEHFLSGDKKAYAKESERLKKEIVKTLIKNLPEGIGKISEINSAKYLNGVLYDFIDKTHKDSEEKQNILSDILETKGYLALFSKFLTSRITTLEQSMPKRVIENFEIYAANIPKMQDALERGAVSFAIEYESICSVDYYNQILSQEDIDSYNRLISGIMDEDGAKEKGINQTISEKNIKIKSEHLEEKPFRILKQLHKQILEEREKAFTIDHIDSDEEVVQVTKEAFEQTKEQWENIKKINGFYAKDPGDITLFIVVGPNQTHVLSQLIYGEHDRIRLLLEEYEKNTLEVLPRRTKSEKARYDKFVNAVPKKVAKESHTFDGLQKMTGDDRLFILYRDELARNYMRIKEAYGTFERDILKSRRGIKGNRDVQESLVSFYDELTKFRSALRIINSGNDEKADPIFYNTFDGIFEKANRTYKAENLCRNYVTKSPADDARIMASCLGTPARLRTHWWNGEENFAINDVAMIRRGDEYYYFVLTPDVKPVDLKTKDETDAQIFVQRKGAKSFLGLPKALFKCILEPYFESPEHKNDKNCVIEEYVSKPLTIDRRAYDIFKNGTFKKTNIGIDGLTEEKFKDDCRYLIDVYKEFIAVYTRYSCFNMSGLKRADEYNDIGEFFSDVDTRLCTMEWIPVSFERINDMVDKKEGLLFLVRSMFLYNRPRKPYERTFIQLFSDSNMEHTSMLLNSRAMIQYRAASLPRRVTHKKGSILVALRDSNGEHIPMHIREAIYKMKNNFDISSEDFIMAKAYLAERDVAIKKANEDIIRNRRYTEDKFFLSLSYTKNADISARTLDYINDKVEEDTQDSRMAVIVTRNLKDLTYVAVVDEKNNVLEEKSLNEIDGVNYRELLKERTKIKYHDKTRLWQYDVSSKGLKEAYVELAVTQISKLATKYNAVVVVESMSSTFKDKFSFLDEQIFKAFEARLCARMSDLSFNTIKEGEAGSISNPIQVSNNNGNSYQDGVIYFLNNAYTRTLCPDTGFVDVFDKTRLITMQSKRQFFAKMKDIRIDDGEMLFTFNLEEYPTKRLLDRKEWTVKIAGDGSYFDKDKGEYVYVNDIVREQIIPALLEDKAVFDGNMAEKFLDKTAISGKSVELIYKWFANALYGIITKKDGEKIYRSPITGTEIDVSKNTTYNFGKKFMFKQEYRGDGDFLDAFLNYMQAQDIAV>AIZ56868_(modified) hypothetical protein Mpt1_c09950 [CandidatusMethanoplasma termitum] (SEQ ID NO: 104)MNNYDEFTKLYPIQKTIRFELKPQGRTMEHLETFNFFEEDRDRAEKYKILKEAIDEYHKKFIDEHLTNMSLDWNSLKQISEKYYKSREEKDKKVFLSEQKRMRQEIVSEFKKDDRFKDLFSKKLFSELLKEEIYKKGNHQEIDALKSFDKFSGYFIGLHENRKNMYSDGDEITAISNRIVNENFPKFLDNLQKYQEARKKYPEWIIKAESALVAHNIKMDEVFSLEYFNKVLNQEGIQRYNLALGGYVTKSGEKMMGLNDALNLAHQSEKSSKGRIHMTPLFKQILSEKESFSYIPDVFTEDSQLLPSIGGFFAQIENDKDGNIFDRALELISSYAEYDTERIYIRQADINRVSNVIFGEWGTLGGLMREYKADSINDINLERTCKKVDKWLDSKEFALSDVLEAIKRTGNNDAFNEYISKMRTAREKIDAARKEMKFISEKISGDEESIHIIKTLLDSVQQFLHFFNLFKARQDIPLDGAFYAEFDEVHSKLFAIVPLYNKVRNYLTKNNLNTKKIKLNFKNPTLANGWDQNKVYDYASLIFLRDGNYYLGIINPKRKKNIKFEQGSGNGPFYRKMVYKQIPGPNKNLPRVFLTSTKGKKEYKPSKEIIEGYEADKHIRGDKFDLDFCHKLIDFFKESIEKHKDWSKFNFYFSPTESYGDISEFYLDVEKQGYRMHFENISAETIDEYVEKGDLFLFQIYNKDFVKAATGKKDMHTIWNAAFSPENLQDVVVKLNGEAELFYRDKSDIKEIVIAREGEILVNRTYNGRTPVPDKIHKKLTDYHNGRTKDLGEAKEYLDKVRYFKAHYDITKDRRYLNDKIYFHVPLTLNFKANGKKNLNKMVIEKFLSDEKAHIIGIDRGERNLLYYSIIDRSGKIIDQQSLNVIDGFDYREKLNQREIEMKDARQSWNAIGKIKDLKEGYLSKAVHEITKMAIQYNAIVVMEELNYGFKRGRFKVEKQIYQKFENMLIDKMNYLVFKDAPDESPGGVLNAYQLTNPLESFAKLGKQTGILFYVPAAYTSKIDPTTGFVNLFNTSSKTNAQERKEFLQKFESISYSAKDGGIFAFAFDYRKFGTSKTDHKNVWTAYTNGERMRYIKEKKRNELFDPSKEIKEALTSSGIKYDGGQNILPDILRSNNNGLIYTMYSSFIAAIQMRVYDGKEDYIISPIKNSKGEFFRTDPKRRELPIDADANGAYNIALRGELTMRAIAEKFDPDSEKMAKLELKHKDWFEFMQTRGD>WP_027407524_(modified) hypothetical protein [Anaerovibrio sp. RM50](SEQ ID NO: 105) MVAFIDEFVGQYPVSKTLRFEARPVPETKKWLESDQCSVLFNDQKRNEYYGVLKELLDDYYRAYIEDALTSFTLDKALLENAYDLYCNRDTNAFSSCCEKLRKDLVKAFGNLKDYLLGSDQLKDLVKLKAKVDAPAGKGKKKIEVDSRLINWLNNNAKYSAEDREKYIKAIESFEGFVTYLTNYKQARENMFSSEDKSTAIAFRVIDQNMVTYFGNIRIYEKIKAKYPELYSALKGFEKFFSPTAYSEILSQSKIDEYNYQCIGRPIDDADFKGVNSLINEYRQKNGIKARELPVMSMLYKQILSDRDNSFMSEVINRNEEAIECAKNGYKVSYALFNELLQLYKKIFTEDNYGNIYVKTQPLTELSQALFGDWSILRNALDNGKYDKDIINLAELEKYFSEYCKVLDADDAAKIQDKFNLKDYFIQKNALDATLPDLDKITQYKPHLDAMLQAIRKYKLFSMYNGRKKMDVPENGIDFSNEFNAIYDKLSEFSILYDRIRNFATKKPYSDEKMKLSFNMPTMLAGWDYNNETANGCFLFIKDGKYFLGVADSKSKNIFDFKKNPIALLDKYSSKDIYYKVKYKQVSGSAKMLPKVVFAGSNEKIFGHLISKRILEIREKKLYTAAAGDRKAVAEWIDFMKSAIAIHPEWNEYFKFKFKNTAEYDNANKFYEDIDKQTYSLEKVEIPTEYIDEMVSQHKLYLFQLYTKDFSDKKKKKGTDNLHTMYWHGVFSDENLKAVTEGTQPIIKLNGEAEMFMRNPSIEFQVTHEHNKPIANKNPLNTKKESVFNYDLIKDKRYTERKFYFHCPITLNFRADKPIKYNEKINRFVENNPDVCIIGIDRGERHLLYYTVINQTGDILEQGSLNKISGSYTNDKGEKVNKETDYHDLLDRKEKGKHVAQQAWETIENIKELKAGYLSQVVYKLTQLMLQYNAVIVLENLNVGFKRGRTKVEKQVYQKFEKAMIDKLNYLVFKDRGYEMNGSYAKGLQLTDKFESFDKIGKQTGCIYYVIPSYTSHIDPKTGFVNLLNAKLRYENITKAQDTIRKFDSISYNAKADYFEFAFDYRSFGVDMARNEWVVCTCGDLRWEYSAKTRETKAYSVTDRLKELFKAHGIDYVGGENLVSHITEVADKHFLSTLLFYLRLVLKMRYTVSGTENENDFILSPVEYAPGKFFDSREATSTEPMNADANGAYHIALKGLMTIRGIEDGKLHNYGKGGENAAWFKFMQNQEY KNNG>WP_044910712_(modified) hypothetical protein [Lachnospiraceae bacteriumMC2017] (SEQ ID NO: 106)MDYGNGQFERRAPLTKTITLRLKPIGETRETIREQKLLEQDAAFRKLVETVTPIVDDORKIADNALCHFGTEYDFSCLGNAISKNDSKAIKKETEKVEKLLAKVLTENLPDGLRKVNDINSAAFIQDTLTSFVQDDADKRVLIQELKGKTVLMQRFLTTRITALTVWLPDRVFENFNIFIENAEKMRILLDSPLNEKIMKFDPDAEQYASLEFYGQCLSQKDIDSYNLIISGIYADDEVKNPGINEIVKEYNQQIRGDKDESPLPKLKKLHKQILMPVEKAFFVRVLSNDSDARSILEKILKDTEMLPSKILEAMKEADAGDIAVYGSRLHELSHVIYGDHGKLSQIIYDKESKRISELMETLSPKERKESKKRLEGLEEHIRKSTYTFDELNRYAEKNVMAAYIAAVEESCAEIMRKEKDLRTLLSKEDVKIRGNRHNTLIVKNYFNAWTVFRNLIRILRRKSEAIDSDFYDVLDDSVEVLSLTYKGENLCRSYITKKIGSDLKPEIATYGSALRPNSRWWSPGEKFNVKFHTIVRRDGRLYYFILPKGAKPVELEDMDGDIECLQMRKIPNPTIFLPKLVFKDPEAFFRDNPEADEFVFLSGMKAPVTITRETYEAYRYKLYTVGKLRDGEVSEEEYKRALLQVLTAYKEFLENRMIYADLNFGFKDLEEYKDSSEFIKQVETHNTFMCWAKVSSSQLDDLVKSGNGLLFEIWSERLESYYKYGNEKVLRGYEGVLLSILKDENLVSMRTLLNSRPMLVYRPKESSKPMVVHRDGSRVVDRFDKDGKYIPPEVHDELYRFFNNLLIKEKLGEKARKILDNKKVKVKVLESERVKWSKFYDEQFAVTFSVKKNADCLDTTKDLNAEVMEQYSESNRLILIRNTTDILYYLVLDKNGKVLKQRSLNIINDGARDVDWKERFRQVTKDRNEGYNEWDYSRTSNDLKEVYLNYALKEIAEAVIEYNAILIIEKMSNAFKDKYSFLDDVTFKGFETKLLAKLSDLHFRGIKDGEPCSFTNPLQLCQNDSNKILQDGVIFMVPNSMTRSLDPDTGFIFAINDHNIRTKKAKLNFLSKFDQLKVSSEGCLIMKYSGDSLPTHNTDNRVWNCCCNHPITNYDRETKKVEFIEEPVEELSRVLEENGIETDTELNKLNERENVPGKVVDAIYSLVLNYLRGTVSGVAGQRAVYYSPVTGKKYDISFIQAMNLNRKCDYYRIGSKERGEWTDFVAQLIN>WP_027216152_(modified) hypothetical protein [Butyrivibrio fibrisolvens](SEQ ID NO: 107) MYYESLTKLYPIKKTIRNELVPIGKTLENIKKNNILEADEDRKIAYIRVKAIMDDYHKRLINEALSGFALIDLDKAANLYLSRSKSADDIESFSRFQDKLRKAIAKRLREHENFGKIGNKDIIPLLQKLSENEDDYNALESFKNFYTYFESYNDVRLNLYSDKEKSSTVAYRLINENLPRFLDNIRAYDAVQKAGITSEELSSEAQDGLFLVNTFNNVLIQDGINTYNEDIGKLNVAINLYNQKNASVQGFRKVPKMKVLYKQILSDREESFIDEFESDTELLDSLESHYANLAKYFGSNKVQLLFTALRESKGVNVYVKNDIAKTSFSNVVFGSWSRIDELINGEYDDNNNRKKDEKYYDKRQKELKKNKSYTIEKIITLSTEDVDVIGKYIEKLESDIDDIRFKGKNFYEAVLCGHDRSKKLSKNKGAVEAIKGYLDSVKDFERDLKLINGSGQELEKNLVVYGEQEAVLSELSGIDSLYNMTRNYLTKKPFSTEKIKLNFNKPTFLDGWDYGNEEAYLGFFMIKEGNYFLAVMDANWNKEFRNIPSVDKSDCYKKVIYKQISSPEKSIQNLMVIDGKTVKKNGRKEKEGIHSGENLILEELKNTYLPKKINDIRKRRSYLNGDTFSKKDLTEFIGYYKQRVIEYYNGYSFYFKSDDDYASFKEFQEDVGRQAYQISYVDVPVSFVDDLINSGKLYLFRVYNKDFSEYSKGRLNLHTLYFKMLFDERNLKNVVYKLNGQAEVFYRPSSIKKEELIVHRAGEEIKNKNPKRAAQKPTRRLDYDIVKDRRYSQDKFMLHTSIIMNFGAEENVSFNDIVNGVLRNEDKVNVIGIDRGERNLLYVVVIDPEGKILEQRSLNCITDSNLDIETDYHRLLDEKESDRKIARRDWTTIENIKELKAGYLSQVVHIVAELVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVMDKSREQLSPEKISGALNALQLTPDFKSFKVLGKQTGIIYYVPAYLTSKIDPMTGFANLFYVKYENVDKAKEFFSKFDSIKYNKDGKNWNTKGYFEFAFDYKKFTDRAYGRVSEWTVCTVGERIIKFKNKEKNNSYDDKVIDLTNSLKELFDSYKVTYESEVDLKDAILAIDDPAFYRDLTRRLQQTLQMRNSSCDGSRDYIISPVKNSKGEFFCSDNNDDTTPNDADANGAFNIARKGLWVLNEIRNSEEGSKINLAMSNAQWLEYAQDNTI>WP_016301126_(modified) hypothetical protein [Lachnospiraceae bacteriumCOE1] (SEQ ID NO: 108)MHEENNGKIADNFIGIYPVSKTLRFELKPVGKTQEYIEKHGILDEDLKRAGDYKSVKKIIDAYHKYFIDEALNGIQLDGLKNYYELYEKKRDNNEEKEFQKIQMSLRKQIVKRFSEHPQYKYLFKKELIKNVLPEFTKDNAEEQTLVKSFQEFTTYFEGFHQNRKNMYSDEEKSTAIAYRVVHQNLPKYIDNMRIFSMILNTDIRSDLTELFNNLKTKMDITIVEEYFAIDGFNKVVNQKGIDVYNTILGAFSTDDNTKIKGLNEYINLYNQKNKAKLPKLKPLFKQILSDRDKISFIPEQFDSDTEVLEAVDMFYNRLLQFVIENEGQITISKLLTNFSAYDLNKIYVKNDTTISAISNDLFDDWSYISKAVRENYDSENVDKNKRAAAYEEKKEKALSKIKMYSIEELNFFVKKYSCNECHIEGYFERRILEILDKMRYAYESCKILEIDKGLINNISLCQDRQAISELKDFLDSIKEVQWLLKPLMIGQEQADKEEAFYTELLRIWEELEPITLLYNKVRNYVTKKPYTLEKVKLNFYKSTLLDGWDKNKEKDNLGIILLKDGQYYLGIMNRRNNKIADDAPLAKTDNVYRKMEYKLLTKVSANLPRIFLKDKYNPSEEMLEKYEKGTHLKGENFCIDDCRELIDFFKKGIKQYEDWGQFDFKFSDTESYDDISAFYKEVEHQGYKITFRDIDETYIDSLVNEGKLYLFQIYNKDFSPYSKGTKNLHTLYWEMLFSQQNLQNIVYKLNGNAEIFYRKASINQKDVVVHKADLPIKNKDPQNSKKESMFDYDIIKDKRFTCDKYQFHVPITMNFKALGENHFNRKVNRLIHDAENMHIIGIDRGERNLIYLCMIDMKGNIVKQISLNEIISYDKNKLEHKRNYHQLLKTREDENKSARQSWQTIHTIKELKEGYLSQVIHVITDLMVEYNAIVVLEDLNFGFKQGRQKFERQVYQKFEKMLIDKLNYLVDKSKGMDEDGGLLHAYQLTDEFKSFKQLGKQSGFLYYIPAWNTSKLDPTTGFVNLFYTKYESVEKSKEFINNFTSILYNQEREYFEFLFDYSAFTSKAEGSRLKWTVCSKGERVETYRNPKKNNEWDTQKIDLTFELKKLFNDYSISLLDGDLREQMGKIDKADFYKKFMKLFALIVQMRNSDEREDKLISPVLNKYGAFFETGKNERMPLDADANGAYNIARKGLWIIEKIKNTDVEQLDKVKLTISNKEWLQYAQEHIL>WP_035635841_(modified) hypothetical protein [Lachnospiraceae bacteriumND2006] (SEQ ID NO: 109)MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKIADDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIIVIYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH>WP_015504779_(modified) exonuclease SbcC [Candidatus Methanomethylophilusalvus] (SEQ ID NO: 110)MDAKEFTGQYPLSKTLRFELRPIGRTWDNLEASGYLAEDRHRAECYPRAKELLDDNHRAFLNRVLPQIDMDWHPIAEAFCKVHKNPGNKELAQDYNLQLSKRRKEISAYLQDADGYKGLFAKPALDEAMKIAKENGNESDIEVLEAFNGFSVYFTGYHESRENIYSDEDMVSVAYRITEDNFPRFVSNALIFDKLNESEEPDIISEVSGNLGVDDIGKYFDVSNYNNFLSQAGIDDYNHIIGGHTTEDGLIQAFNVVLNLRHQKDPGFEKIQFKQLYKQILSVRTSKSYIPKQFDNSKEMVDCICDYVSKIEKSETVERALKLVRNISSFDLRGIFVNKKNLRILSNKLIGDWDAIETALMHSSSSENDKKSVYDSAEAFTLDDIFSSVKKFSDASAEDIGNRAEDICRVISETAPFINDLRAVDLDSLNDDGYEAAVSKIRESLEPYMDLFHELEIFSVGDEFPKCAAFYSELEEVSEQLIEIIPLFNKARSFCTRKRYSTDKIKVNLKFPTLADGWDLNKERDNKAAILRKDGKYYLAILDMKKDLSSIRTSDEDESSFEKMEYKLLPSPVKMLPKIFVKSKAAKEKYGLTDRMLECYDKGMHKSGSAFDLGFCHELIDYYKRCIAEYPGWDVFDFKFRETSDYGSMKEFNEDVAGAGYYMSLRKIPCSEVYRLLDEKSIYLFQIYNKDYSENAHGNKNMHTMYWEGLFSPQNLESPVFKLSGGAELFFRKSSIPNDAKTVHPKGSVLVPRNDVNGRRIPDSIYRELTRYFNRGDCRISDEAKSYLDKVKTKKADHDIVKDRRFTVDKMMFHVPIAMNFKAISKPNLNKKVIDGIIDDQDLKIIGIDRGERNLIYVTMVDRKGNILYQDSLNILNGYDYRKALDVREYDNKEARRNWTKVEGIRKMKEGYLSLAVSKLADMIIENNAIIVMEDLNHGFKAGRSKIEKQVYQKFESMLINKLGYMVLKDKSIDQSGGALHGYQLANHVTTLASVGKQCGVIFYIPAAFTSKIDPTTGFADLFALSNVKNVASMREFFSKMKSVIYDKAEGKFAFTFDYLDYNVKSECGRTLWTVYTVGERFTYSRVNREYVRKVPTDIIYDALQKAGISVEGDLRDRIAESDGDTLKSIFYAFKYALDMRVENREEDYIQSPVKNASGEFFCSKNAGKSLPQDSDANGAYNIALKGILQLRMLSEQYDPNAESIRLPLITNKAWLTFMQSGMKTWKN>WP_044910713_(modified) hypothetical protein [Lachnospiraceae bacteriumMC2017] (SEQ ID NO: 111)MGLYDGFVNRYSVSKTLRFELIPQGRTREYIETNGILSDDEERAKDYKTIKRLIDEYHKDYISRCLKNVNISCLEEYYHLYNSSNRDKRHEELDALSDQMRGEIASFLTGNDEYKEQKSRDIIINERIINFASTDEELAAVKRFRKFTSYFTGFFTNRENMYSAEKKSTAIAHRIIDVNLPKYVDNIKAFNTAIEAGVFDIAEFESNFKAITDEREVSDLLDITKYSRFIRNEDIIIYNTLLGGISMKDEKIQGLNELINLHNQKHPGKKVPLLKVLYKQILGDSQTHSFVDDQFEDDQQVINAVKAVTDTFSETLLGSLKIIINNIGHYDLDRIYIKAGQDITTLSKRALNDWHIITECLESEYDDKFPKNKKSDTYEEMRNRYVKSFKSFSIGRLNSLVTTYTEQACFLENYLGSFGGDTDKNCLTDFTNSLMEVEHLLNSEYPVTNRLITDYESVRILKRLLDSEMEVIHFLKPLLGNGNESDKDLVFYGEFEAEYEKLLPVIKVYNRVRNYLTRKPFSTEKIKLNFNSPTLLCGWSQSKEKEYMGVILRKDGQYYLGIIVITPSNKKIFSEAPKPDEDCYEKMVLRYIPHPYQMLPKVFFSKSNIAFFNPSDEILRIKKQESFKKGKSFNRDDCHKFIDFYKDSINRHEEWRKFNFKFSDTDSYEDISRFYKEVENQAFSMSFTKIPTVYIDSLVDEGKLYLFKLHNKDFSEHSKGKPNLHTVYWNALFSEYNLQNTVYQLNGSAEIFFRKASIPENERVIHKKNVPITRKVAELNGKKEVSVFPYDIIKNRRYTVDKFQFHVPLKMNFKADEKKRINDDVIEAIRSNKGIHVIGIDRGERNLLYLSLINEEGRIIEQRSLNIIDSGEGHTQNYRDLLDSREKDREKARENWQEIQEIKDLKTGYLSQAIHTITKWMKEYNAIIVLEDLNDRFTNGRKKVEKQVYQKFEKMLIDKLNYYVDKDEEFDRMGGTHRALQLTEKFESFQKLGRQTGFIFYVPAWNTSKLDPTTGFVDLLYPKYKSVDATKDFIKKFDFIRFNSEKNYFEFGLHYSNFTERAIGCRDEWILCSYGNRIVNFRNAAKNNSWDYKEIDITKQLLDLFEKNGIDVKQENLIDSICEMKDKPFFKSLIANIKLILQIRNSASGTDIDYMISPAMNDRGEFFDTRKGLQQLPLDADANGAYNIAKKGLWIVDQIRNTTGNNVKMAMSNREWMHFAQESRLA >KKQ36153_(modified) hypothetical protein US52_C0007G0008 [candidatedivision WS6 bacterium GW2011_GWA2_37_6] (SEQ ID NO: 112)MKNVFGGFTNLYSLTKTLRFELKPTSKTQKLMKRNNVIQTDEEIDKLYHDEMKPILDEIHRRFINDALAQKIFISASLDNFLKVVKNYKVESAKKNIKQNQVKLLQKEITIKTLGLRREVVSGFITVSKKWKDKYVGLGIKLKGDGYKVLTEQAVLDILKIEFPNKAKYIDKFRGFWTYFSGFNENRKNYYSEEDKATSIANRIVNENLSRYIDNIIAFEEILQKIPNLKKFKQDLDITSYNYYLNQAGIDKYNKIIGGYIVDKDKKIQGINEKVNLYTQQTKKKLPKLKFLFKQIGSERKGFGIFEIKEGKEWEQLGDLFKLQRTKINSNGREKGLFDSLRTMYREFFDEIKRDSNSQARYSLDKIYFNKASVNTISNSWFTNWNKFAELLNIKEDKKNGEKKIPEQISIEDIKDSLSIIPKENLEELFKLTNREKHDRTRFFGSNAWVTFLNIWQNEIEESFNKLEEKEKDFKKNAAIKFQKNNLVQKNYIKEVCDRMLAIERMAKYHLPKDSNLSREEDFYWIIDNLSEQREIYKYYNAFRNYISKKPYNKSKMKLNFENGNLLGGWSDGQERNKAGVILRNGNKYYLGVLINRGIFRTDKINNEIYRTGSSKWERLILSNLKFQTLAGKGFLGKHGVSYGNMNPEKSVPSLQKFIRENYLKKYPQLTEVSNTKFLSKKDFDAAIKEALKECFTMNFINIAENKLLEAEDKGDLYLFEITNKDFSGKKSGKDNIHTIYWKYLFSESNCKSPIIGLNGGAEIFFREGQKDKLHTKLDKKGKKVFDAKRYSEDKLFFHVSITINYGKPKNIKFRDIINQLITSMNVNIIGIDRGEKHLLYYSVIDSNGIILKQGSLNKIRVGDKEVDFNKKLTERANEMKKARQSWEQIGNIKNFKEGYLSQAIHEIYQLMIKYNAIIVLEDLNTEFKAKRLSKVEKSVYKKFELKLARKLNHLILKDRNTNEIGGVLKAYQLTPTIGGGDVSKFEKAKQWGMMFYVRANYTSTTDPVTGWRKHLYISNFSNNSVIKSFFDPTNRDTGIEIFYSGKYRSWGFRYVQKETGKKWELFATKELERFKYNQTTKLCEKINLYDKFEELFKGIDKSADIYSQLCNVLDFRWKSLVYLWNLLNQIRNVDKNAEGNKNDFIQSPVYPFFDSRKTDGKTEPINGDANGALNIARKGLMLVERIKNNPEKYEQLIRDTEWDAWIQNFNKVN>WP_044919442_(modified) hypothetical protein [Lachnospiraceae bacteriumMA2020] (SEQ ID NO: 113)MYYESLTKQYPVSKTIRNELIPIGKTLDNIRQNNILESDVKRKQNYEHVKGILDEYHKQLINEALDNCTLPSLKIAAEIYLKNQKEVSDREDFNKTQDLLRKEVVEKLKAHENFTKIGKKDILDLLEKLPSISEDDYNALESFRNFYTYFTSYNKVRENLYSDKEKSSTVAYRLINENFPKFLDNVKSYRFVKTAGILADGLGEEEQDSLFIVETFNKTLTQDGIDTYNSQVGKINSSINLYNQKNQKANGFRKIPKMKMLYKQILSDREESFIDEFQSDEVIIDNVESYGSVLIESLKSSKVSAFFDALRESKGKNVYVKNDLAKTAMSNIVFENWRTFDDLLNQEYDLANENKKKDDKYFEKRQKELKKNKSYSLEHLCNLSEDSCNLIENYIHQISDDIENIIINNETFLRIVINEHDRSRKLAKNRKAVKAIKDFLDSIKVLERELKLINSSGQELEKDLIVYSAHEELLVELKQVDSLYNMTRNYLTKKPFSTEKVKLNFNRSTLLNGWDRNKETDNLGVLLLKDGKYYLGIMNTSANKAFVNPPVAKTEKVFKKVDYKLLPVPNQMLPKVFFAKSNIDFYNPSSEIYSNYKKGTHKKGNMFSLEDCHNLIDFFKESISKREDWSKFGFKFSDTASYNDISEFYREVEKQGYKLTYTDIDETYINDLIERNELYLFQIYNKDFSMYSKGKLNLHTLYFMMLFDQRNIDDVVYKLNGEAEVFYRPASISEDELIIHKAGEEIKNKNPNRARTKETSTFSYDIVKDKRYSKDKFTLHIPITMNFGVDEVKRFNDAVNSAIRIDENVNVIGIDRGERNLLYVVVIDSKGNILEQISLNSIINKEYDIETDYHALLDEREGGRDKARKDWNTVENIRDLKAGYLSQVVNVVAKLVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVIDKSREQTSPKELGGALNALQLTSKFKSFKELGKQSGVIYYVPAYLTSKIDPTTGFANLFYMKCENVEKSKRFFDGFDFIRFNALENVFEFGFDYRSFTQRACGINSKWTVCTNGERIIKYRNPDKNNMFDEKVVVVTDEMKNLFEQYKIPYEDGRNVKDMIISNEEAEFYRRLYRLLQQTLQMRNSTSDGTRDYIISPVKNKREAYFNSELSDGSVPKDADANGAYNIARKGLWVLEQIRQKSEGEKINLAMTNAEWLEY AQTHLL>WP_035798880_(modified) hypothetical protein [Butyrivibrio sp. NC3005](SEQ ID NO: 114) MYYQNLTKKYPVSKTIRNELIPIGKTLENIRKNNILESDVKRKQDYEHVKGIMDEYHKQLINEALDNYMLPSLNQAAEIYLKKHVDVEDREEFKKTQDLLRREVTGRLKEHENYTKIGKKDILDLLEKLPSISEEDYNALESFRNFYTYFTSYNKVRENLYSDEEKSSTVAYRLINENLPKFLDNIKSYAFVKAAGVLADCIEEEEQDALFMVETFNMTLTQEGIDMYNYQIGKVNSAINLYNQKNHKVEEFKKIPKMKVLYKQILSDREEVFIGEFKDDETLLSSIGAYGNVLMTYLKSEKINIFFDALRESEGKNVYVKNDLSKTTMSNIVFGSWSAFDELLNQEYDLANENKKKDDKYFEKRQKELKKNKSYTLEQMSNLSKEDISPIENYIERISEDIEKICIYNGEFEKIVVNEHDSSRKLSKNIKAVKVIKDYLDSIKELEHDIKLINGSGQELEKNLVVYVGQEEALEQLRPVDSLYNLTRNYLTKKPFSTEKVKLNFNKSTLLNGWDKNKETDNLGILFFKDGKYYLGIMNTTANKAFVNPPAAKTENVFKKVDYKLLPGSNKMLPKVFFAKSNIGYYNPSTELYSNYKKGTHKKGPSFSIDDCHNLIDFFKESIKKHEDWSKFGFEFSDTADYRDISEFYREVEKQGYKLTFTDIDESYINDLIEKNELYLFQIYNKDFSEYSKGKLNLHTLYFMMLFDQRNLDNVVYKLNGEAEVFYRPASIAENELVIHKAGEGIKNKNPNRAKVKETSTFSYDIVKDKRYSKYKFTLHIPITMNFGVDEVRRFNDVINNALRTDDNVNVIGIDRGERNLLYVVVINSEGKILEQISLNSIINKEYDLETNYHALLDEREDDRNKARKDWNTLENIKELKTGYLSQVVNVVAKLVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIEKLNYLVIDKSREQVSPEKMGGALNALQLTSKFKSFAELGKQSGIIYYVPAYLTSKIDPTTGFVNLFYIKYENIEKAKQFFDGFDFIRFNKKDDMFEFSFDYKSFTQKACGIRSKWIVYTNGERIIKYPNPEKNNLFDEKVINVTDEIKGLFKQYRIPYENGEDIKEIIISKAEADFYKRLFRLLHQTLQMRNSTSDGTRDYIISPVKNDRGEFFCSEFSEGTMPKDADANGAYNIARKGLWVLEQIRQKDEGEKVNLSMTNAEWLKYAQLHLL>WP_027109509_(modified) hypothetical protein [Lachnospiraceae bacterium NC2008](SEQ ID NO: 1581) MENYYDSLTRQYPVTKTIRQELKPVGKTLENIKNAEIIEADKQKKEAYVKVKELMDEFHKSIIEKSLVGIKLDGLSEFEKLYKIKTKTDEDKNRISELFYYMRKQIADALKNSRDYGYVDNKDLIEKILPERVKDENSLNALSCFKGFTTYFTDYYKNRKNIYSDEEKHSTVGYRCINENLLIFMSNIEVYQIYKKANIKNDNYDEETLDKTFMIESFNECLTQSGVEAYNSVVASIKTATNLYIQKNNKEENFVRVPKMKVLFKQILSDRTSLFDGLIIESDDELLDKLCSFSAEVDKFLPINIDRYIKTLMDSNNGTGIYVKNDSSLTTLSNYLTDSWSSIRNAFNENYDAKYTGKVNDKYEEKREKAYKSNDSFELNYIQNLLGINVIDKYIERINFDIKEICEAYKEMTKNCFEDEEDKTKKLQKNIKAVASIKSYLDSLKNIERDIKLLNGTGLESRNEFFYGEQSTVLEEITKVDELYNITRNYLTKKPFSTEKMKLNFNNPQLLGGWDVNKERDCYGVILIKDNNYYLGIMDKSANKSFLNIKESKNENAYKKVNCKLLPGPNKMFPKVFFAKSNIDYYDPTHEIKKLYDKGTFKKGNSFNLEDCHKLIDFYKESIKKNDDWKNFNFNFSDTKDYEDISGFFREVEAQNYKITYTNVSCDFIESLVDEGKLYLFQIYNKDFSEYATGNLNLHTLYLKMLFDERNLKDLCIKMNGEAEVFYRPASILDEDKVVHKANQKITNKNTNSKKKESIFSYDIVKDKRYTVDKFFIHLPITLNYKEQNVSRFNDYIREILKKSKNIRVIGIDRGERNLLYVVVCDSDGSILYQRSINEIVSGSHKTDYHKLLDNKEKERLSSRRDWKTIENIKDLKAGYMSQVVNEIYNLILKYNAIVVLEDLNIGFKNGRKKVEKQVYQNFEKALIDKLNYLCIDKTREQLSPSSPGGVLNAYQLTAKFESFEKIGKQTGCIFYVPAYLTSQIDPTTGFVNLFYQKDTSKQGLQLFFRKFKKINFDKVASNFEFVFDYNDFTNKAEGTKTNWTISTQGTRIAKYRSDDANGKWISRTVHPTDIIKEALNREKINYNDGHDLIDEIVSIEKSAVLKEIYYGFKLTLQLRNSTLANEEEQEDYIISPVKNSSGNYFDSRITSKELPCDADANGAYNIARKGLWALEQIRNSENVSKVKLAISNKEWFEYTQNNIPSL>WP_029202018_(modified) hypothetical protein [Oribacterium sp. NK2B42](SEQ ID NO: 115) MYYDGLTKQYALSKTIRNELVPIGKTLDNIKKNRILEADIKRKSDYEHVKKLMDMYHKKIINEALDNFKLSVLEDAADIYFNKQNDERDIDAFLKIQDKLRKEIVEQLKGHTDYSKVGNKDFLGLLKAASTEEDRILIESFDNFYTYFTSYNKVRSNLYSAEDKSSTVAYRLINENLPKFFDNIKAYRTVRNAGVISGDMSIVEQDELFEVDTFNHTLTQYGIDTYNHMIGQLNSAINLYNQKMHGAGSFKKLPKMKELYKQLLTEREEEFIEEYTDDEVLITSVHNYVSYLIDYLNSDKVESFFDTLRKSDGKEVFIKNDVSKTTMSNILFDNWSTIDDLINHEYDSAPENVKKTKDDKYFEKRQKDLKKNKSYSLSKIAALCRDTTILEKYIRRLVDDIEKIYTSNNVFSDIVLSKHDRSKKLSKNTNAVQAIKNMLDSIKDFEHDVMLINGSGQEIKKNLNVYSEQEALAGILRQVDHIYNLTRNYLTKKPFSTEKIKLNFNRPTFLDGWDKNKEEANLGILLIKDNRYYLGIMNTSSNKAFVNPPKAISNDIYKKVDYKLLPGPNKMLPKVFFATKNIAYYAPSEELLSKYRKGTHKKGDSFSIDDCRNLIDFFKSSINKNTDWSTFGFNFSDTNSYNDISDFYREVEKQGYKLSFTDIDACYIKDLVDNNELYLFQIYNKDFSPYSKGKLNLHTLYFKMLFDQRNLDNVVYKLNGEAEVFYRPASIESDEQIIHKSGQNIKNKNQKRSNCKKTSTFDYDIVKDRRYCKDKFMLHLPITVNFGTNESGKFNELVNNAIRADKDVNVIGIDRGERNLLYVVVVDPCGKIIEQISLNTIVDKEYDIETDYHQLLDEKEGSRDKARKDWNTIENIKELKEGYLSQVVNIIAKLVLKYDAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDKMNYLVLDKSRKQESPQKPGGALNALQLTSAFKSFKELGKQTGIIYYVPAYLTSKIDPTTGFANLFYIKYESVDKARDFFSKFDFIRYNQMDNYFEFGFDYKSFTERASGCKSKWIACTNGERIVKYRNSDKNNSFDDKTVILTDEYRSLFDKYLQNYIDEDDLKDQILQIDSADFYKNLIKLFQLTLQMRNSSSDGKRDYIISPVKNYREEFFCSEFSDDTFPRDADANGAYNIARKGLWVIKQIRETKSGTKINLAMSNSEWLEYAQCNLL>WP_028248456_(modified) hypothetical protein [Pseudobutyrivibrio ruminis](SEQ ID NO: 116) MYYQNLTKMYPISKTLRNELIPVGKTLENIRKNGILEADIQRKADYEHVKKLMDNYHKQLINEALQGVHLSDLSDAYDLYFNLSKEKNSVDAFSKCQDKLRKEIVSLLKNHENFPKIGNKEIIKLLQSLYDNDTDYKALDSFSNFYTYFSSYNEVRKNLYSDEEKSSTVAYRLINENLPKFLDNIKAYAIAKKAGVRAEGLSEEDQDCLFIIETFERTLTQDGIDNYNAAIGKLNTAINLFNQQNKKQEGFRKVPQMKCLYKQILSDREEAFIDEFSDDEDLITNIESFAENMNVFLNSEIITDFKIALVESDGSLVYIKNDVSKTSFSNIVFGSWNAIDEKLSDEYDLANSKKKKDEKYYEKRQKELKKNKSYDLETIIGLFDDNSDVIGKYIEKLESDITAIAEAKNDFDEIVLRKHDKNKSLRKNTNAVEAIKSYLDTVKDFERDIKLINGSGQEVEKNLVVYAEQENILAEIKNVDSLYNMSRNYLTQKPFSTEKFKLNFNRATLLNGWDKNKETDNLGILFEKDGMYYLGIMNTKANKIFVNIPKATSNDVYHKVNYKLLPGPNKMLPKVFFAQSNLDYYKPSEELLAKYKAGTHKKGDNFSLEDCHALIDFFKASIEKHPDWSSFGFEFSETCTYEDLSGFYREVEKQGYKITYTDVDADYITSLVERDELYLFQIYNKDFSPYSKGNLNLHTIYLQMLFDQRNLNNVVYKLNGEAEVFYRPASINDEEVIIHKAGEEIKNKNSKRAVDKPTSKFGYDIIKDRRYSKDKFMLHIPVTMNFGVDETRRFNDVVNDALRNDEKVRVIGIDRGERNLLYVVVVDTDGTILEQISLNSIINNEYSIETDYHKLLDEKEGDRDRARKNWTTIENIKELKEGYLSQVVNVIAKLVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVIDKSRKQDKPEEFGGALNALQLTSKFTSFKDMGKQTGIIYYVPAYLTSKIDPTTGFANLFYVKYENVEKAKEFFSRFDSISYNNESGYFEFAFDYKKFTDRACGARSQWTVCTYGERIIKFRNTEKNNSFDDKTIVLSEEFKELFSIYGISYEDGAELKNKIMSVDEADFFRSLTRLFQQTMQMRNSSNDVTRDYIISPIMNDRGEFFNSEACDASKPKDADANGAFNIARKGLWVLEQIRNTPSGDKLNLAMSNAEWLEYAQRNQI>WP_028830240_(modified) hypothetical protein [Proteocatella sphenisci](SEQ ID NO: 117) MENFKNLYPINKTLRFELRPYGKTLENFKKSGLLEKDAFKANSRRSMQAIIDEKFKETIEERLKYTEFSECDLGNMTSKDKKITDKAATNLKKQVILSFDDEIFNNYLKPDKNIDALFKNDPSNPVISTFKGFTTYFVNFFEIRKHIFKGESSGSMAYRIIDENLTTYLNNIEKIKKLPEELKSQLEGIDQIDKLNNYNEFITQSGITHYNEIIGGISKSENVKIQGINEGINLYCQKNKVKLPRLTPLYKMILSDRVSNSFVLDTLENDTELIEMISDLINKTEISQDVIMSDIQNIFIKYKQLGNLPGISYSSIVNAICSDYDNNFGDGKRKKSYENDRKKHLETNVYSINYISELLTDTDVSSNIKMRYKELEQNYQVCKENFNATNWMNIKNIKQSEKTNIIKDLLDILKSIQRFYDLFDIVDEDKNPSAEFYTWLSKNAEKLDFEFNSVYNKSRNYLTRKQYSDKKIKLNFDSPTLAKGWDANKEIDNSTIIMRKFNNDRGDYDYFLGIWNKSTPANEKIIPLEDNGLFEKMQYKLYPDPSKMLPKQFLSKIWKAKHPTTPEFDKKYKEGRHKKGPDFEKEFLHELIDCFKHGLVNHDEKYQDVFGFNLRNTEDYNSYTEFLEDVERCNYNLSFNKIADTSNLINDGKLYVFQIWSKDFSIDSKGTKNLNTIYFESLFSEENMIEKMFKLSGEAEIFYRPASLNYCEDIIKKGHHHAELKDKFDYPIIKDKRYSQDKFFFHVPMVINYKSEKLNSKSLNNRTNENLGQFTHIIGIDRGERHLIYLTVVDVSTGEIVEQKHLDEIINTDTKGVEHKTHYLNKLEEKSKTRDNERKSWEAIETIKELKEGYISHVINEIQKLQEKYNALIVMENLNYGFKNSRIKVEKQVYQKFETALIKKFNYIIDKKDPETYIHGYQLTNPITTLDKIGNQSGIVLYIPAWNTSKIDPVTGFVNLLYADDLKYKNQEQAKSFIQKIDNIYFENGEFKFDIDFSKWNNRYSISKTKWTLTSYGTRIQTFRNPQKNNKWDSAEYDLTEEFKLILNIDGTLKSQDVETYKKFMSLFKLMLQLRNSVTGTDIDYMISPVTDKTGTHFDSRENIKNLPADADANGAYNIARKGIMAIENIMNGISDPLKISNEDYLKYIQNQQE

Applicants generated vector constructs as shown in FIGS. 40A-L (e.g.PACYC184 fnCpf1 (PY001)) and FIGS. 41A-E (e.g. PaCpf1).

PAM Challenge Assay for detection of putative PAM sequences for FnCpf1(FIG. 42 ): Applicants isolated the Cpf1 loci from Francisella novicida(Fn) (FIG. 43 ) and transformed it into E. coli. The locus was expressedin E. coli from pACYC184 similar to the experiment described inSapranauskas et al.

E. coli with pACYC-FnCpf1 locus=Cpf1+E. coli with empty pACYC184=control

Applicants transformed Cpf1+ and control E. coli with PAM libraryplasmids. Two PAM libraries were obtained (FIG. 44 ). PAM libraries arepUC19 plasmids containing a 31 bp proto-spacer sequence which matchesspacer 1 in FnCpf1 locus. PAM left library had a 8 nt degenerate PAM atthe 5′ end of the proto-spacer. PAM right library had a 7 nt degeneratePAM at the 3′ end of the proto-spacer. Applicants plated Cpf1+ andcontrol E. coli and harvested all colonies after ˜12 h. Each colonyrepresented a PAM-pUC19 transformation event that did not result incutting/interference by Cpf1. These PAM-pUC19 plasmids do not carry arecognizable PAM. Applicants determined from sequencing of all colonieswhich PAM-pUC19 plasmids were no longer present compared to control andthese plasmids were identified to contain a recognizable PAM.

Cloning of pY0001: pY0001 is a pACYC184 backbone (from NEB) with apartial FnCpf1 locus. pY0001 contains the endogenous FnCpf1 locus from255 bp of the acetyltransferase 3′ sequence to the 4th spacer sequence.Only spacer 1-3 are potentially active since spacer 4 is not longerflanked by direct repeats.

Applicants PCR amplified the FnCpf1 locus in 3 pieces and cloned intoXba1 & Hind3 cut pACYC184 using Gibson assembly.

Cpf1 PAM Screen Computational Analysis

After sequencing of the screen DNA, Applicants extracted the regionscorresponding to either the left PAM or the right PAM. For each sample,the number of PAMs present in the sequenced library were compared to thenumber of expected PAMs in the library (4{circumflex over ( )}8 for theleft library, 4{circumflex over ( )}7 for the right).The left library showed PAM depletion. To quantify this depletion,Applicants calculated an enrichment ratio. For both conditions (controlpACYC or FnCpf1 containing pACYC), Applicants calculated the ratio foreach PAM in the library as:

${ratio} = {{- \log_{2}}\frac{{sample} + 0.01}{{{initial}\mspace{14mu}{library}} + 0.01}}$

Applicants determined that plotting the distribution showed littleenrichment in the control sample and enrichment in both bioreps.Applicants collected all PAMs above a ratio of 8, and plotted thefrequency distributions, revealing a 5′ YYN PAM (FIGS. 45A-E).Applicants confirmed that the PAM is TTN, where N is A/C/G or T.

Applicants performed RNA-sequencing on Francisella tolerances Cpf1 locusand the RNAseq analysis showed that the CRISPR locus was activelyexpressed (FIG. 46 ). A further depiction of the RNAseq analysis of theFnCpf1 locus is shown in FIG. 86 . In addition to the Cpf1 and Casgenes, two small non-coding transcripts were highly transcribed, whichApplicants surmised were putative tracrRNAs. The CRISPR array is alsoexpressed. Both the putative tracrRNAs and CRISPR array are transcribedin the same direction as the Cpf1 and Cas genes. Here all RNAtranscripts identified through the RNAseq experiment are mapped againstthe locus. Zooming into the Cpf1 CRISPR array Applicants identified manydifferent short transcripts. In this plot, all identified RNAtranscripts are mapped against the Cpf1 locus (FIG. 47 ). Afterselecting transcripts that are less than 85 nucleotides long, Applicantsidentified two putative tracrRNAs (FIG. 48 ). FIG. 49 shows a zoomed inperspective of putative tracrRNA 1 and the CRISPR array. FIG. 50 shows azoomed in perspective of putative tracrRNA 2. Putative crRNA sequencesare indicated in FIG. 51 .

Applicants test for function in mammalian cells using U6 PCR products:spacer (DR-spacer-DR) (in certain aspects spacers may be referred to ascrRNA or guide RNA or an analogous term as described in thisapplication) and tracr for other identified Cpf1 loci.

Example 4: Further Validation Experiments for FnCpf1

Applicants confirmed the predicted FnCpf1 PAM is TTN in vivo by usingthe assay outlined in FIG. 52 . Applicants transformed FnCpf1 locuscarrying cells and control cells with pUC19 encoding endogenous spacer 1with 5′ TTN PAM (FIG. 53 ). Briefly, in the in vivo PAM confirmationassay, 50 μl of competent E. coli with FnCpf1 locus (test strain) orwith empty pACYC184 (control strain) were transformed with 10 ngproto-spacer 1 carrying plasmids. Preceding the proto-spacer sequenceare predicted PAM sequences (TTC, TTG, TTA and TTT). Aftertransformation cells were diluted 1:2000 and plated on LB agar platescontaining ampicillin and chloramphenicol. Only cells with intactproto-spacer plasmid can form colonies. Plates with colonies were imaged˜14 h after plating and colonies were counted using the ImageJ software.

Applicants performed Cell Lysate Cleavage Assays to further validateFnCpf1 cleavage. The protocol for the cell lysate cleavage assay is asfollows:

In vitro cleavage reaction. Cleavage buffer: 100 mM HEPES pH 7.5, 500 mMKCl, 25 mM MgCl2, 5 mM DTT, 25% glycerol. The stock may be made withoutDTT.

Making Cell Lysate

Lysis buffer: 20 mM Hepes pH 7.5, 100 mM potassium chloride [KCl], 5 mMmagnesium chloride [MgCl2], 1 mM dithiothreitol [DTT], 5% glycerol, 0.1%Triton X-100, supplemented with 10× Roche Protease Inhibitor cocktail.Concentrated stock of lysis buffer w/o Roche Protease Inhibitor and DTTmay be maintained. Keep at −20° C.

Transfect HEK cells with recommended amount of DNA with Lipofectamine2000

500 ng per 24 well

2000 ng per 6 well

Harvest cells with lysis buffer 24-72 hours post transfection

Aspirate off media

Wash gently with DPBS

Aspirate off DPBS

Use 50 ul of lysis buffer per 24 well or 250 ul per 6 well

Let sit on ice for 5 min

Transfer into Eppendorf tube

Ice for 15 minutes

Sonicate at high power, 50% duty cycle for 5-10 min

Spin down cold at max speed for 20 min

Transfer supernatant to new tube

Aliquot in PCR strip tubes, 10 ul per strip and freeze at −80C

In vitro transcription of guide RNA

Kit protocol: Information may be accessed at the websitewww.neb.com/products/e2030-hiscribe-t7-in-vitro-transcription-kit

Take 100 uM stock oligoAnneal in 10 ul reaction:1 ul of T7 “forward” strand=“XRP2649”1 ul of T7 “reverse” oligo1 ul TaqB buffer7 ul water

Run the PNK PCR program without the 37° C. incubation step (basicallyheat up to 95° C. for 5 min and do slow cool to 4° C. but not as slow assurveyor anneal). Nanodrop annealed oligos: normalize with water to 500ng/ul (usually 1000-2000 ng/ul for a 120 nt oligo)

For T7 transcription follow kit instructions (but cut down size by 4×)

10 ul Reaction

1 ul 10× buffer1 ul T7 transcriptase0.5 ul rNTP

0.5 ul HMW mix

1 ul DNA template (annealed)6 ul water

Transcribe in 42° C. (preferably thermocycler) for at least 2-3 hours,let run overnight. Yield should be around 1000-2000 ng/ul of RNA. It isnormal for white residues to form.

Preparation of DNA

For pUC19, linearize with HindIII and column purify

will need 300-400 ng of plasmid per reaction, so cut amount necessary

For gDNA, amplify wt cell DNA with PCR

do several PCR reactions, pool and column purify

concentrate the product so around 100-200 ng/ul

Keep at −20 C

20 ul reaction

10 ul of lysate (this is pre-aliquoted)2 ul of cleavage buffer (NEB buffer 3)1 ul of RNA (directly from above; don't need to purify)1 ul of DNA (from above)6 ul of waterIncubate at 37° C. for 1-2 hour (30 min is enough)

Column purify the reaction

Run out on a 2% E-gel

The cell lysate cleavage assay used tracrRNA at positions 1, 2, 3, 4 and5 as indicated in FIG. 54 . Cell Lysate Cleavage Assay (1) (FIG. 55 ) isa gel indicating the PCR fragment with a TTa PAM and proto-spacer1sequence incubated in cell lysate. Cell Lysate Cleavage Assay (2) (FIG.56 ) is a gel showing the pUC-spacer1 with different PAMs incubated incell lysate. Cell Lysate Cleavage Assay (3) (FIG. 57 ) is a gel showingthe BasI digestion after incubation in cell lysate. Cell Lysate CleavageAssay (4) (FIG. 58 ) is a gel showing digestion results for threeputative crRNA sequences.

Applicants also determined the effect of spacer length on cleavageefficiency. Applicants tested different lengths of spacer against apiece of target DNA containing the target site:5′-TTAgagaagtcatttaataaggccactgttaaaa-3′ (SEQ ID NO: 119). For thisexperiment, pUC19 plasmid containing the spacer(5′-TTcgagaagucauuuaauaaggccacuguuaaaa-3′ (SEQ ID NO: 120)) was treatedto the following conditions:

2 ul cell lysate containing Cpf1 2 ul pUC19 DNA with spacer (300 ng) 1ul crRNA (500 ng) 2 ul NEBuffer 3 2 ul 40 mM DTT 0.3 ul BsaI 10.7 ulddH2O

Incubated at 37 C for 30 minutes, followed by treatment with RNase for 5minutes. Then the reaction was cleaned up using Qiagen PCR PurificationKit and analyzed on 2% Invitrogen E-gel EX. FIG. 59 is a gel showingthat crRNAs 1-7 mediated successful cleavage of the target DNA in vitrowith FnCpf1, whereas crRNAs 8-13 did not facilitate cleavage of thetarget DNA.

Applicants arrived at the minimal Fn Cpf1 locus (FIG. 60 ) and alsoelucidated the minimal Cpf1 guide (FIG. 61 ). Applicants also cleaved aPCR amplicon of the human Emx1 locus (FIG. 81 ). The EMX amplicon wastreated to the following conditions:

2 ul cell lysate containing Cpf1 3 ul pUC19 DNA with spacer (300 ng) 1ul crRNA (500 ng) 2 ul NEBuffer 3 2 ul 40 mM DTT 0.3 ul BsaI 9.7 ulddH₂O

Incubated at 37° C. for 30 minutes, followed by treatment with RNase for5 minutes. Then the reaction was cleaned up using Qiagen PCRPurification Kit and analyzed on 2% Invitrogen E-gel EX.

Applicants further studied the effect of truncation in 5′ DR on cleavageactivity (FIG. 82A-B). For this experiment, pUC19 plasmid containing thespacer (5′-TTcgagaagucauuuaauaaggccacuguuaaaa-3′ (SEQ ID NO: 121)) wastreated to the following conditions:

2 ul cell lysate containing Cpf1 2 ul pUC19 DNA with spacer (300 ng) 1ul crRNA (500 ng) 2 ul NEBuffer 3 2 ul 40 mM DTT 0.3 ul BsaI 10.7 ulddH2O

Incubated at 37° C. for 30 minutes, followed by treatment with RNase for5 minutes. Then the reaction was cleaned up using Qiagen PCRPurification Kit and analyzed on 2% Invitrogen E-gel EX. Applicantsdetermined that crDNA deltaDR5 disrupted the stem loop at the 5′ end andthis shows that the stemloop at the 5′ end is essential for cleavageactivity (FIG. 82B).

Applicants investigated the effect of crRNA-DNA target mismatch oncleavage efficiency (FIG. 83 ). For this experiment, pUC19 plasmidcontaining the spacer (5′-TTcgagaagucauuuaauaaggccacuguuaaaa-3′ (SEQ IDNO: 122)) was treated to the following conditions:

2 ul cell lysate containing Cpf1 2 ul pUC19 DNA with spacer (300 ng) 1ul crRNA (500 ng) 2 ul NEBuffer 3 2 ul 40 mM DTT 0.3 ul BsaI 10.7 ulddH2O

Incubated at 37 C for 30 minutes, followed by treatment with RNase for 5minutes. Then the reaction was cleaned up using Qiagen PCR PurificationKit and analyzed on 2% Invitrogen E-gel EX. Each lane in the gel shownin FIG. 83 consists of Cpf1-containing cell lysate, pUC19 with TTcprotospacer, and the corresponding crRNA, indicated as 1-11.

Applicants studied the FnCpf1p RuvC domain and have identified aminoacid mutations that may convert the FnCpf1 effector protein into anickase, whereby the effector protein has substantially reduced nucleaseactivity and only one strand of DNA is nicked and/or cleaved. The aminoacid positions in the FnCpf1p RuvC domain include but are not limited toD917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A,D1227A, D1255A and N1257A. The amino acid positions in AsCpf1 correspondto AsD908A, AsE993A, AsD1263A. The amino acid positions in LbCpf1correspond to LbD832A

Applicants have also identified a putative second nuclease domain whichis most similar to PD-(D/E)XK nuclease superfamily and HincIIendonuclease like. The point mutations to be generated in this putativenuclease domain to substantially reduce nuclease activity include butare not limited to N580A, N584A, T587A, W609A, D610A, K613A, E614A,D616A, K624A, D625A, K627A and Y629A.

Applicants perform plasmid cleavage experiments with FnCpf1p andsequencing of said plasmids will provide information as to whether thecut site is sticky or blunt. Applicants will elucidate further detailson the various domains of FnCpf1p from the crystal structure of thisprotein in a suitable complex. For optimization of FnCpf1 locicomponents for activity in human cells, Applicants will try differentarchitectures of crRNAs and try more targets than described herein.

Applicants cleaved DNA using purified Francisella and Prevotella Cpf1(FIG. 84 ). For this experiment, pUC19 plasmid containing the spacer(5′-TTcgagaagucauuuaauaaggccacuguuaaaa-3′ (SEQ ID NO: 123)) was treatedto the following conditions:

2 ul purified protein solution 2 ul pUC19 DNA with spacer (300 ng) 1 ulcrRNA (500 ng) 2 ul NEBuffer 3 2 ul 40 mM DTT 0.3 ul BsaI 10.7 ul ddH2O

Incubated at 37° C. for 30 minutes, followed by treatment with RNase for5 minutes. Then the reaction was cleaned up using Qiagen PCRPurification Kit and analyzed on 2% Invitrogen E-gel EX. Analysis of thegel shown in FIG. 84 indicates that PaCpf1 can work with FnCpf1 crRNA,although the activity is not as high as FnCpf1. Applicants concludedthat this makes sense given the stem-loop sequences for PaCpf1 andFnCpf1 are almost identical (only 1 base difference) (see FIGS. 85A-B).This is further highlighted in the mature crRNA sequences for FnCpf1 andPaCpf1 shown in FIGS. 87A-B. In preferred embodiments of the invention,biochemical or in vitro cleavage may not require a tracr sequence foreffective function of a Cpf1p CRISPR system. Inclusion of a stem loop ora further optimized stem loop structure is important for cleavageactivity.

DNA cleavage by human codon optimized Francisella novicida FnCpf1p.

Applicants also showed that FnCpf1p cleaves DNA in human cells. 400 nghuman codon optimized FnCpf1p and 100 ng U6::crRNA were transfected perwell of HEK293T cells (˜240,000 cells) in 24 well plates. Five crRNAscomprising spacer sequences of length 20-24 nt based on5′-ctgatggtccatgtctgttactcg-3′ (SEQ ID NO: 124) (i.e., the first 20, 21,22, 23, or all 24 nt) were employed. The crRNAs further comprised 20 ntof the 5′ repeat sequence of PaCpf1 at the 5′ of the spacer. Applicantsearlier determined that the repeat sequence from PaCpf1 can berecognized by FnCpf1.

DNA was harvested after ˜60 h and analyzed by SURVEYOR nuclease assay.The SURVEYOR primers for DNMT1 were 5′-ctgggactcaggcgggtcac-3′ (SEQ IDNO: 125) (forward) and 5′-cctcacacaacagcttcatgtcagc-3′ (SEQ ID NO: 126)(reverse). Cleaved DNA fragments coinciding with expected cleavageproducts of ˜345 bp and ˜261 bp were observed for all five crRNAs(spacer lengths 20-24 nt). (FIG. 88 ).

Example 5: Further Validation Experiments for PaCpf1

A PAM computational screen was performed for Prevotella albensisCpf1(PaCpf1) similar to the screen performed for FnCpf1 as detailed inExample 3. After sequencing of the screen DNA, the regions correspondingto either the left PAM or the right PAM were extracted.

For each sample, the number of PAMs present in the sequenced librarywere compared to the number of expected PAMs in the library(4{circumflex over ( )}7). The left library showed very slight PAMdepletion. To quantify this depletion, an enrichment ratio wascalculated. For both conditions (control pACYC or PaCpf1 containingpACYC) the ratio was calculated for each PAM in the library as

${ratio} = {{- \log_{2}}\frac{{sample} + 0.01}{{{initial}\mspace{14mu}{library}} + 0.01}}$

Plotting the distribution shows little enrichment in the control sampleand enrichment in both bioreps. All PAMs above a ratio of 4.5 werecollected, and the frequency distributions were plotted, revealing a 5′TTTV PAM, where V is A or C or G (FIG. 62A-E).

Applicants will elucidate further details on the various domains ofPaCpf1p from the crystal structure of this protein in a suitablecomplex. For optimization of PaCpf1 loci components for activity inhuman cells, Applicants will work with different crRNA (guideRNA)architectures and different optimized PaCpf1 effector proteins.Applicants have human codon optimized the PaCpf1 sequence as follows:

NLS (underline)

GS linker (bold)

3xHA tag (italics)

(SEQ ID NO: 127) ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAG CCggtagtAACATCAAAAACTTTACCGGGCTCTACCCCCTCAGCAAAACTTTGCGCTTTGAACTCAAGCCTATTGGCAAAACCAAGGAAAACATCGAGAAAAATGGCATCCTGACCAAGGACGAGCAACGGGCTAAAGACTACCTCATAGTCAAAGGCTTTATTGACGAGTATCACAAGCAGTTCATCAAAGACAGGCTTTGGGACTTTAAATTGCCTCTCGAAAGTGAGGGGGAGAAGAACAGTCTCGAAGAATACCAGGAACTGTACGAGCTCACTAAGCGCAACGATGCCCAGGAGGCCGACTTCACCGAGATTAAAGATAACCTTCGCAGCTCTATTACCGAACAGCTCACGAAGTCTGGATCTGCGTACGATCGGATTTTTAAAAAAGAGTTCATTAGAGAAGACCTGGTCAACTTCCTCGAAGATGAAAAAGATAAAAATATCGTGAAACAGTTCGAGGACTTTACTACATATTTTACGGGTTTTTATGAAAATAGGAAGAACATGTACTCTAGCGAAGAGAAGTCCACGGCCATCGCATACCGGCTTATCCATCAGAATCTGCCAAAATTCATGGACAACATGAGAAGTTTTGCCAAAATTGCAAATTCCAGTGTTTCCGAGCACTTTAGCGACATCTATGAAAGCTGGAAGGAATATCTGAATGTAAATAGCATCGAGGAAATCTTCCAGCTCGACTATTTTAGCGAAACCTTGACTCAGCCACATATTGAGGTGTATAACTATATTATCGGGAAGAAAGTCCTGGAAGACGGAACCGAGATAAAGGGCATCAACGAGTATGTGAACCTCTACAATCAGCAGCAGAAAGATAAGAGTAAACGACTGCCTTTCCTGGTGCCACTGTATAAGCAAATTTTGTCTGATAGGGAAAAACTCTCCTGGATTGCTGAAGAGTTCGACAGCGACAAGAAGATGCTGAGCGCTATCACCGAGTCTTACAACCACCTGCACAACGTGTTGATGGGTAACGAGAACGAAAGCCTGCGAAATCTGCTGCTGAATATTAAGGACTATAACCTGGAGAAAATTAATATCACAAACGACTTGTCTCTCACCGAAATCTCCCAGAATCTTTTTGGCCGATATGATGTATTCACAAATGGGATCAAAAACAAGCTGAGAGTGTTGACTCCAAGGAAGAAAAAGGAGACGGACGAAAATTTTGAGGACCGCATTAACAAAATTTTTAAGACCCAGAAGTCCTTCAGCATCGCTTTTCTGAACAAGCTGCCTCAGCCCGAAATGGAGGATGGGAAGCCCCGGAACATTGAGGACTATTTCATTACACAGGGGGCGATTAACACCAAATCTATACAGAAAGAAGATATCTTCGCCCAAATTGAGAATGCATACGAGGATGCACAGGTGTTCCTGCAAATTAAGGACACCGACAACAAACTTAGCCAGAACAAGACGGCGGTGGAAAAGATCAAAACTTTGCTGGACGCCTTGAAGGAACTCCAGCACTTCATCAAACCGCTGCTGGGCTCTGGGGAGGAGAACGAGAAAGACGAACTGTTCTACGGTTCCTTCCTGGCCATCTGGGACGAACTGGACACCATTACACCACTTTATAACAAAGTGAGAAATTGGCTGACCCGAAAACCATATTCAACAGAAAAAATCAAATTGAATTTCGACAACGCTCAGCTGCTGGGAGGGTGGGATGTCAATAAAGAACACGACTGTGCAGGTATCTTGTTGCGGAAAAACGATAGCTACTATCTCGGAATTATCAATAAGAAAACCAACCACATCTTTGATACGGATATTACGCCATCAGATGGCGAGTGCTATGACAAAATCGACTACAAGCTCCTTCCCGGGGCGAACAAAATGCTTCCAAAGGTGTTTTTTAGTAAGTCCCGAATCAAAGAGTTCGAGCCATCAGAGGCCATAATCAATTGCTATAAGAAGGGGACACACAAAAAAGGAAAAAACTTTAACCTGACGGACTGTCACCGCCTGATCAACTTTTTTAAGACCTCAATCGAGAAACACGAGGATTGGTCAAAATTCGGATTCAAGTTCTCCGATACCGAAACGTATGAGGATATTAGCGGTTTTTATAGAGAGGTCGAGCAGCAGGGATACAGGCTGACGAGCCATCCAGTCAGTGCCAGCTATATACATAGTCTGGTCAAGGAAGGAAAACTGTACCTCTTCCAAATCTGGAACAAGGACTTTTCTCAATTCTCCAAGGGGACCCCTAACTTGCACACTCTCTATTGGAAGATGCTGTTTGACAAACGGAATCTTAGCGATGTGGTTTATAAGCTGAATGGCCAGGCTGAAGTGTTCTATAGAAAGAGCTCCATTGAACACCAGAACCGAATTATCCACCCCGCTCAGCATCCCATCACAAATAAGAATGAGCTTAACAAAAAGCACACTAGCACCTTCAAATACGATATCATCAAAGATCGCAGATACACGGTGGATAAATTCCAGTTCCATGTGCCCATTACTATAAATTTTAAGGCGACCGGGCAGAACAACATCAACCCAATCGTCCAAGAGGTGATTCGCCAAAACGGTATCACCCACATCATAGGCATCGATCGAGGTGAACGCCATCTTCTGTACCTCTCTCTCATCGATTTGAAAGGCAACATCATCAAGCAGATGACTCTCAACGAAATTATTAATGAGTATAAGGGTGTGACCTATAAGACCAACTACCATAACCTCCTGGAGAAGAGGGAGAAGGAGCGGACCGAGGCCAGACACTCCTGGAGTAGTATTGAAAGCATAAAAGAACTGAAGGATGGATACATGTCACAGGTGATTCACAAAATTACGGACATGATGGTTAAGTACAATGCGATTGTGGTCCTGGAGGACCTCAACGGGGGGTTTATGCGAGGCCGCCAGAAGGTCGAGAAGCAGGTGTACCAGAAATTTGAAAAAAAGTTGATCGACAAGCTGAACTATCTCGTTGACAAGAAACTCGACGCTAACGAGGTCGGCGGAGTACTGAATGCTTATCAGCTGACCAACAAGTTCGAGTCTTTCAAGAAGATTGGGAAACAAAGCGGATTTTTGTTCTACATCCCCGCCTGGAACACAAGCAAAATCGATCCTATAACAGGGTTCGTTAATCTGTTCAACACCAGGTACGAGTCTATCAAGGAGACAAAAGTTTTTTGGTCTAAGTTTGATATTATCCGATACAATAAAGAGAAGAATTGGTTCGAGTTCGTCTTCGATTACAATACCTTTACGACTAAAGCGGAGGGAACACGCACTAAGTGGACTCTGTGCACCCACGGCACTCGCATCCAGACATTCCGGAACCCAGAAAAGAATGCCCAGTGGGACAATAAAGAGATCAATTTGACTGAGTCCTTCAAAGCTCTGTTTGAAAAGTACAAGATCGATATCACCAGTAATCTCAAGGAATCCATCATGCAGGAAACCGAGAAGAAGTTCTTCCAGGAACTGCATAATCTGCTCCACCTGACCCTGCAGATGAGGAATAGCGTTACTGGAACCGACATAGACTATTTGATCAGCCCCGTTGCCGATGAGGATGGAAATTTCTATGATAGTCGCATAAATGGCAAAAATTTTCCGGAGAATGCCGATGCCAATGGCGCGTACAACATCGCACGAAAGGGTCTGATGCTTATTCGGCAGATCAAGCAAGCAGATCCACAGAAGAAATTCAAGTTTGAGACAATCACCAATAAAGACTGGCTGAAATTCGCCCAAGACAAGCCCTATCTTAAAGATggcagcggg AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG ggatcc TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATACGATGTCCCCGACT ATGCCTAA

The vector map for human codon optimized PaCpf1 sequence is provided inFIG. 63 .

Example 6: Cpf1 Orthologs

Applicants analyzed an expanding pool of Cpf1 orthologs (FIG. 64 ).Human codon optimized sequences were obtained for several Cpf1 locicomponents (FIGS. 65-79 ). Applicants also arrived at the Direct Repeat(DR) sequences for each ortholog and their predicted fold structure(FIG. 80A-I).

Applicants further study Cpf1 orthologs based on size of the effectorprotein, i.e. smaller effector proteins allow for easier packaging intovectors and on PAM composition. All aspects allow for furtheroptimization in prokaryotic and eukaryotic cells, preferably foreffective activity in mammalian cells, i.e. human cells.

Applicants showed that the effector protein orthologs of the followingloci showed activity in the in vitro cleavage assay: Peregrinibacteriabacterium GW2011_GWA2_33_10 Cpf1, Acidaminococcus sp. BV3L6 Cpf1,Francisalla tularensis 1 Cpf1, Moraxella bovoculi 237 Cpf1,Lachnospiraceae bacterium ND2006 Cpf1, Lachnospiraceaa bacterium MA2020Cpf1, Porphyromonas macacee Cpf1, Porphyromonas crevlorlcanls 3 Cpf1,Prevotella albensis Cpf1 (FIG. 64 ).

In the in vitro cleavage assay by orthologs, HEK293 cells expressingCpf1 orthologs were harvested and the lysate was incubated withpredicted mature crRNA targeting an artificial spacer cloned into thepUC19 plasmids. The spacer was preceded by 8 degenerate bases to allowfor determination of the PAM via sequencing. The lower bands signifycleavage by the Cpf1 enzyme (FIG. 89 ).

Applicants identified computationally derived PAMs from the in vitrocleavage assay (FIG. 90 ). Uncut DNA from FIG. 89 (the higher band) wasexcised and amplified for next generation sequencing. The abundance ofeach 8-mer was calculated and the log ratio compared to the inputlibrary was used to quantify enrichment. Individual 8-mers with a logratio greater than 4 were compiled and used to determine the consensusPAM using Weblogo.

Applicants further identified that Cpf1p effector proteins cut in astaggered fashion with 5′ overhangs. Purified FnCpf1 protein washarvested and incubated with crRNA and the corresponding target clonedinto pUC19. The cleaved product was gel extracted and submitted forSanger sequencing. The asymmetric reads show that there is a staggeredcut (FIG. 91 ). In a preferred embodiment of the invention, Applicantsdemonstrate in vivo staggered ligation with a template (e.g. anexogenous template).

Applicants also determined the effect of spacer length on the cuttingability of the effector protein (FIG. 92 ). Purified FnCpf1 protein washarvested and incubated with crRNA and the corresponding target clonedinto pUC19. Spacer lengths greater than 17 nt cut to completion, whilethe 17 nt spacer shows reduced activity and spacers less than 17 nt arenot active.

Applicants demonstrated that FnCpf1 mediates indel formation in HEK293Tcells.

˜280,000 HEK cells/24 well were transfected with 350 ng of huFnCpf1plasmid and 150 ng U6::crRNA. Cells were harvested three days aftertransfection and analyzed by SURVEYOR nuclease assay. Uncleaved PCRfragment size is 606 bps. Expected fragment sizes are ˜418 bp and ˜188bp for crRNA DNMT1-1 and ˜362 bp and ˜244 bp for crRNA DNMT1-3 (FIG. 93).

(SEQ ID NO: 128) DNMT1-1 spacer sequence: cctcactcctgctcggtgaattt(SEQ ID NO: 129) DNMT1-3 spacer sequence: ctgatggtccatgtctgttactc

Applicants identified the required components of the Cpf1 system toachieve cleaveage by determining if transcripts were processed whencertain sequences of the locus were deleted (FIG. 94A-F). The deletedsequences may include but are not limited to the Cas1 gene, the Cas2gene and the tracr. Hence, in a preferred embodiment of the invention,Applicants demonstrated that the tracr is not a required component of afunctional Cpf1 system or complex to achieve cleavage.

Example 7: Procedures Generation of Heterologous Plasmids

To generate the FnCpf1 locus for heterologous expression, genomic DNAfrom Francisella Novicida was PCR amplified using Herculase IIpolymerase (Agilent Technologies) and cloned into pACYC-184 using Gibsoncloning (New England Biolabs). Cells harboring plasmids were madecompetent using the Z-competent kit (Zymo).

Bacterial RNA-Sequencing

RNA was isolated from stationary phase bacteria by first resuspending F.novicida (generous gift from David Weiss) or E. coli in TRIzol and thenhomogenizing the bacteria with zirconia/silica beads (BioSpec Products)in a BeadBeater (BioSpec Products) for 3 one-minute cycles. Total RNAwas purified from homogenized samples with the Direct-Zol RNA miniprepprotocol (Zymo), DNase treated with TURBO DNase (Life Technologies), and3′ dephosphorylated with T4 Polynucleotide Kinase (New England Biolabs).rRNA was removed with the bacterial Ribo-Zero rRNA removal kit(Illumina). RNA libraries were prepared from rRNA-depleted RNA usingNEBNext® Small RNA Library Prep Set for Illumina (New England Biolabs)and size selected using the Pippin Prep (Sage Science)

For heterologous E. coli expression of the FnCpf1 locus, RNA sequencinglibraries were prepared from rRNA-depleted RNA using a derivative of thepreviously described CRISPR RNA sequencing method (Heidrich et al.,2015. Briefly, transcripts were poly-A tailed with E. coli Poly(A)Polymerase (New England Biolabs), ligated with 5′ RNA adapters using T4RNA Ligase 1 (ssRNA Ligase) High Concentration (New England Biolabs),and reverse transcribed with AffinityScript Multiple Temperature ReverseTranscriptase (Agilent Technologies). cDNA was PCR amplified withbarcoded primers using Herculase II polymerase (Agilent Technologies)RNA-sequencing analysis

The prepared cDNA libraries were sequenced on a MiSeq (Illumina). Readsfrom each sample were identified on the basis of their associatedbarcode and aligned to the appropriate RefSeq reference genome using BWA(Li and Durbin, 2009). Paired-end alignments were used to extract entiretranscript sequences using Picard tools(http://broadinstitute.github.io/picard), and these sequences wereanalyzed using Geneious 8.1.5.

In Vivo FnCpf1 PAM Screen

Randomized PAM plasmid libraries were constructed using synthesizedoligonucleotides (IDT) consisting of 7 randomized nucleotides eitherupstream or downstream of the spacer 1 target (Supplementary Table S8).The randomized ssDNA oligos were made double stranded by annealing to ashort primer and using the large Klenow fragment (New England Biolabs)for second strand synthesis. The dsDNA product was assembled into alinearized pUC19 using Gibson cloning (New England Biolabs). CompetentStbl3 E. coli (Invitrogen) were transformed with the cloned products,and more than 10⁷ cells were collected and pooled. Plasmid DNA washarvested using a Maxi-prep kit (Qiagen). We transformed 360 ng of thepooled library into E. coli cells carrying the FnCpf1 locus or pACYC184control. After transformation, cells were plated on ampicillin. After 16hours of growth, >4*10⁶ cells were harvested and plasmid DNA wasextracted using a Maxi-prep kit (Qiagen). The target PAM region wasamplified and sequenced using a MiSeq (Illumina) with single-end 150cycles.

Computational PAM Discovery Pipeline

PAM regions were extracted, counted, and normalized to total reads foreach sample. For a given PAM, enrichment was measured as the log ratiocompared to pACYC184 control, with a 0.01 psuedocount adjustment. PAMsabove a 3.5 enrichment threshold were collected and used to generatesequence logos (Crooks et al., 2004).

PAM Validation

Sequences corresponding to both PAMs non-PAMs were cloned into digestedpUC19 and ligated with T4 ligase (Enzymatics). Competent E. coli witheither the FnCpf1 locus plasmid or pACYC184 control plasmid weretransformed with 20 ng of PAM plasmid and plated on LB agar platessupplemented with ampicillin and chloramphenicol. Colonies were countedafter 18 hours.

Synthesis of crRNAs and gRNAs

All crRNA and gRNAs used in vitro were synthesized using the HiScribe™T7 High Yield RNA Synthesis Kit (NEB). ssDNA oligos corresponding to thereverse complement of the target RNA sequence were synthesized from IDTand annealed to a short T7 priming sequence. T7 transcription wasperformed for 4 hours and then RNA was purified using the MEGAclear™Transcription Clean-Up Kit (Ambion).

Purification of Cpf1 Protein

FnCpf1 protein was cloned into a bacterial expression vector(6-His-MBP-TEV-Cpf1, a pET based vector kindly given to Applicants byDoug Daniels) (“6-His” disclosed as SEQ ID NO: 130). Two liters ofTerrific Broth growth media with 100 μg/mL ampicillin was inoculatedwith 10 mL overnight culture Rosetta (DE3) pLyseS (EMD Millipore) cellscontaining the Cpf1 expression construct. Growth media plus inoculantwas grown at 37° C. until the cell density reached 0.2 OD600, then thetemperature was decreased to 21° C. Growth was continued until OD600reached 0.6 when a final concentration of 500 μM IPTG was added toinduce MBP-Cpf1 expression. The culture was induced for 14-18 hoursbefore harvesting cells and freezing at −80° C. until purification.

Cell paste was resuspended in 200 mL of Lysis Buffer (50 mM Hepes pH 7,2M NaCl, 5 mM MgCl₂, 20 mM imidazole) supplemented with proteaseinhibitors (Roche cOmplete, EDTA-free) and lysozyme. Once homogenized,cells were lysed by sonication (Branson Sonifier 450) then centrifugedat 10,000 g for 1 hour to clear the lysate. The lysate was filteredthrough 0.22 micron filters (Millipore, Stericup) and applied to anickel column (HisTrap FF, 5 mL), washed, and then eluted with agradient of imidazole. Fractions containing protein of the expected sizewere pooled, TEV protease (Sigma) was added, and the sample was dialyzedovernight into TEV buffer (500 mM NaCl, 50 mM Hepes pH 7, 5 mM MgCl, 2mM DTT). After dialysis, TEV cleavage was confirmed by SDS-PAGE, and thesample was concentrated to 500 μL prior to loading on a gel filtrationcolumn (HiLoad 16/600 Superdex 200) via FPLC (AKTA Pure). Fractions fromgel filtration were analyzed by SDS-PAGE; fractions containing Cpf1 werepooled and concentrated to 200 μL and either used directly forbiochemical assays or frozen at −80° C. for storage. Gel filtrationstandards were run on the same column equilibrated in 2M NaCl, Hepes pH7.0 to calculate the approximate size of FnCpf1.

Generation of Cpf1 Protein Lysate

Cpf1 proteins codon optimized for human expression were synthesized withan N-terminal nuclear localization tag and cloned into the pcDNA3.1expression plasmid by Genscript. 2000 ng of Cpf1 expression plasmidswere transfected into 6-well plates of HEK293FT cells at 90% confluencyusing Lipofectamine 2000 reagent (Life Technologies). 48 hours later,cells were harvested by washing once with DPBS (Life Technologies) andscraping in lysis buffer [20 mM Hepes pH 7.5, 100 mM KCl, 5 mM MgCl2, 1mM DTT, 5% glycerol, 0.1% Triton X-100, 1× cOmplete Protease InhibitorCocktail Tablets (Roche)]. Lysate was sonicated for 10 minutes in aBiorupter sonicator (Diagenode) and then centrifuged. Supernatant wasfrozen for subsequent use in in vitro cleavage assays.

In Vitro Cleavage Assay

Cleavage in vitro was performed either with purified protein ormammalian lysate with protein at 37° C. in cleavage buffer (NEBuffer 3,5 mM DTT) for 20 minutes. The cleavage reaction used 500 ng ofsynthesized crRNA or sgRNA and 200 ng of target DNA. Target DNA involvedeither protospacers cloned into pUC19 or PCR amplicons of gene regionsfrom genomic DNA isolated from HEK293 cells. Reactions were cleaned upusing PCR purification columns (Qiagen) and run on 2% agarose E-gels(Life Technologies). For native and denaturing gels to analyze cleavageby nuclease mutants, cleaned-up reactions were run on TBE 6%polyacrylamide or TBE-Urea 6% polyacrylamide gels (Life Technologies)

In Vitro Cpf1-Family Protein PAM Screen

In vitro cleavage reactions with Cpf1-family proteins were run on 2%agarose E-gels (Life Technologies). Bands corresponding to un-cleavedtarget were gel extracted using QIAquick Gel Extraction Kit (Qiagen) andthe target PAM region was amplified and sequenced using a MiSeq(Illumina) with single-end 150 cycles. Sequencing results were enteredinto the PAM discovery pipeline.

Activity of Cpf1 Cleavage in 293FT Cells

Cpf1 proteins codon optimized for human expression were synthesized withan N-terminal nuclear localization tag and cloned into the pcDNA3.1 CMVexpression plasmid by Genscript. PCR amplicons comprised of a U6promoter driving expression of the crRNA sequence were generated usingHerculase II (Agilent Technologies). 400 ng of Cpf1 expression plasmidsand 100 ng of the crRNA PCR products were transfected into 24-wellplates of HEK293FT cells at 75-90% confluency using Lipofectamine 2000reagent (Life Technologies). Genomic DNA was harvested usingQuickExtract™ DNA Extraction Solution (Epicentre). SURVEYOR nucleaseassay for genome modification

293FT cells were transfected with 400 ng Cpf1 expression plasmid and 100ng U6::crRNA PCRfragments using Lipofectamin 2000 reagent (LifeTechnologies). Cells were incubated at 37° C. for 72 h post-transfectionbefore genomic DNA extraction. Genomic DNA was extracted using theQuickExtract DNA Extraction Solution (Epicentre) following themanufacturer's protocol. The genomic region flanking the CRISPR targetsite for each gene was PCR amplified, and products were purified usingQiaQuick Spin Column (Qiagen) following the manufacturer's protocol.200-500 ng total of the purified PCR products were mixed with 1 μl 10×Taq DNA Polymerase PCR buffer (Enzymatics) and ultrapure water to afinal volume of 10 and subjected to a re-annealing process to enableheteroduplex formation: 95° C. for 10 min, 95° C. to 85° C. ramping at−2° C./s, 85° C. to 25° C. at −0.25° C./s, and 25° C. hold for 1 min.After reannealing, products were treated with SURVEYOR nuclease andSURVEYOR enhancer S (Integrated DNA Technologies) following themanufacturer's recommended protocol, and analyzed on 4-20% Novex TBEpolyacrylamide gels (Life Technologies). Gels were stained with SYBRGold DNA stain (Life Technologies) for 10 min and imaged with a Gel Docgel imaging system (Bio-rad). Quantification was based on relative bandintensities. Indel percentage was determined by the formula,100×(1−(1−(b+c)/(a+b+c))½), where a is the integrated intensity of theundigested PCR product, and b and c are the integrated intensities ofeach cleavage product.

Deep Sequencing to Characterize Cpf1 Indel Patterns in 293FT Cells

HEK293FT cells were transfected and harvested as described for assessingactivity of Cpf1 cleavage. The genomic region flanking DNMT1 targetswere amplified using a two-round PCR region to add Illumina P5 adaptersas well as unique sample-specific barcodes to the target amplicons. PCRproducts were ran on 2% E-gel (Invitrogen) and gel-extracted usingQiaQuick Spin Column (Qiagen) as per the manufacturer's recommendedprotocol. Samples were pooled and quantified by Qubit 2.0 Fluorometer(Life Technologies). The prepared cDNA libraries were sequenced on aMiSeq (Illumina). Indels were mapped using a Python implementation ofthe Geneious 6.0.3 Read Mapper.

Computational Analysis of Cpf1 Loci

PSI-BLAST program (Altschul et al., 1997) was used to identify Cpf1homologs in the NCBI NR database using several known Cpf1 sequences asqueries with the Cpf1 with the E-value cut-off of 0.01 and lowcomplexity filtering and composition based statistics turned off. TheTBLASTN program with the E-value cut-off of 0.01 and low complexityfiltering turned off parameters was used to search the NCBI WGS databaseusing the Cpf1 profile (Marakova et al., 2015) as the query. Results ofall searches were combined. The HHpred program was used with defaultparameters to identify remote sequence similarity using a subset ofrepresentative Cpf1 sequences queries (Soding et al., 2006). Multiplesequence alignment were constructed using MUSCLE (Edgar, 2004) withmanual correction based on pairwise alignments obtained using PSI-BLASTand HHpred programs. Phylogenetic analysis was performed using theFastTree program with the WAG evolutionary model and the discrete gammamodel with 20 rate categories (Price et al., 2010). Protein secondarystructure was predicted using Jpred 4 (Drozdetskiy et al., 2015).

CRISPR repeats were identified using PILER-CR (Edgar, 2007) andCRISPRfinder (Grissa et al, 2007). The spacer sequences were searchedagainst the NCBI nucleotide NR databases using MEGABLAST (Morgulis etal, 2008) with default parameters except that the word size was set at20 and E-value cutoff 0.0001.

TABLE 1 Endogenous F. novicida spacer sequences Spacer number Sequence 1GAGAAGTCATTTAATAAGGCCACTGTTAAAA (SEQ ID NO: 131) 2GCTACTATTCCTGTGCCTTCAGATAATTCA (SEQ ID NO: 132) 3GTCTAGAGCCTTTTGTATTAGTAGCCG (SEQ ID NO: 133)

TABLE 2 ssDNA oligos and primer for generation of PAM libraryOligo/primer name Sequence PAM library 5′ (+)GGCCAGTGAATTCGAGCTCGGTACCCGGG NNNNNNNNGAGAAGTCATTTAATAAGGCCACTGTTAAAAAGCTTGGCGTAATCATGG TCATAGCTGTTT (SEQ ID NO: 134)PAM library 3′ (+) GGCCAGTGAATTCGAGCTCGGTACCCGGGGAGAAGTCATTTAATAAGGCCACTGTTAA AANNNNNNNNAGCTTGGCGTAATCATGGTCATAGCTGTTT (SEQ ID NO: 135) PAM library (−)GCTGACATGAAGCTGTTGTGTGAGG (SEQ ID NO: 136)

TABLE 3 Primers used for pUC19 sequencing and SURVEYOR assay Primer nameSequence NGS pUC For GGCCAGTGAATTCGAGCTCGG (SEQ ID NO: 137) NGS pUC RevCAATTTCACACAGGAAACAGCTATGACC (SEQ ID NO: 138) Sanger pUC ForCGGGGCTGGCTTAACTATGCG (SEQ ID NO: 139) Sanger pUC RevGCCCAATACGCAAACCGCCT (SEQ ID NO: 140) EMX1 ForCCATCCCCTTCTGTGAATGT (SEQ ID NO: 141) EMX1 RevTCTCCGTGTCTCCAATCTCC (SEQ ID NO: 142) DNMT1 ForCTGGGACTCAGGCGGGTCAC (SEQ ID NO: 143) DNMT1 RevGCTGACATGAAGCTGTTGTGTGAGG (SEQ ID NO: 144)

TABLE 4 Truncated guides for in vitro cleavage assay Truncated guidenumber Sequence 1 GAGAAGTCATTTAATAAGGCCACT (SEQ ID NO: 145) 2GAGAAGTCATTTAATAAGGCCA (SEQ ID NO: 146) 3 GAGAAGTCATTTAATAAGGC (SEQ IDNO: 147) 4 GAGAAGTCATTTAATAAG (SEQ ID NO: 148) 5GAGAAGTCATTTAATAA (SEQ ID NO: 149) 6 GAGAAGTCATTTAATA (SEQ ID NO: 150)

TABLE 5 Mismatched guides for in vitro cleavage assay Mismatchedguide number Sequence 1 GATAAGTCATTTAATAAGGCCACT (SEQ ID NO: 151) 2GAGAAGGCATTTAATAAGGCCACT (SEQ ID NO: 152) 3 GAGAAGTCATGTAATAAGGCCACT(SEQ ID NO: 153) 4 GAGAAGTCATTTAAGAAGGCCACT (SEQ ID NO: 154) 5GAGAAGTCATTTAATAAGTCCACT (SEQ ID NO: 155) 6 GAGAAGTCATTTAATAAGGCCAAT(SEQ ID NO: 156)

TABLE 6 Truncated direct repeat guides for in vitro cleavage assayDirect repeat length Sequence +18ATTTCTACTGTTGTAGATGAGAAGTCATTTAATAAGGCC ACT (SEQ ID NO: 157) +17TTTCTACTGTTGTAGATGAGAAGTCATTTAATAAGGCCA CT (SEQ ID NO: 158) +16TTCTACTGTTGTAGATGAGAAGTCATTTAATAAGGCCAC T (SEQ ID NO: 159) +15TCTACTGTTGTAGATGAGAAGTCATTTAATAAGGCCACT (SEQ ID NO: 160) +11CTGTTGTAGATGAGAAGTCATTTAATAAGGCCACT (SEQ ID NO: 161) +7TGTAGATGAGAAGTCATTTAATAAGGCCACT (SEQ ID NO: 162)

TABLE 7 Direct repeat stem mutations for in vitro cleavage assayDirect repeat stem mutant number Sequence 1AATTTCTGCTGTTGCAGAT (SEQ ID NO: 163) 2 AATTTCCACTGTTGTGGAT (SEQ ID NO:164) 3 AATTCCTACTGTTGTAGGT(SEQ ID NO: 165) 4AATTTATACTGTTGTAGAT(SEQ ID NO: 166) 5 AATTTCGACTGTTGTAGATAATTTCGACTGTTGTAGAT (SEQ ID NO: 167) 6 AATTTCTAGTGTTGTAGAT (SEQ ID NO:168)

TABLE 8 Direct repeat loop mutations for in vitro cleavage assayDirect repeat loop mutant number Sequence 1 AATTTCTACTATTGTAGAT (SEQ IDNO: 169) 2 AATTTCTACTGCTGTAGAT (SEQ ID NO: 170) 3AATTTCTACTTTGTAGAT (SEQ ID NO: 171) 4 AATTTCTACTTGTAGAT (SEQ ID NO: 172)5 AATTTCTACTTTTGTAGA (SEQ ID NO: 173) 6 AATTTCTACTTTTGTAGAC (SEQ IDNO: 174)

TABLE 9 Ortholog specific DNMT1 targeting guides for mammalian cellsNuclease Name 5′ Direct Repeat Sequence AsCpf1 DNMT1 target 15′ Direct Repeat Sequence AsCpf1 DNMT1 target 2 TAATTTCTACTGTTGTAGATCCTCACTCCTGC (SEQ ID NO: 175) TCGGTGAATTT (SEQ ID NO: 176) AsCpf1DNMT1 target 3 TAATTTCTACTGTTGTAGAT AGGAGTGTTCAG (SEQ ID NO: 177)TCTCCGTGAAC (SEQ ID NO: 178) AsCpf1 DNMT1 target 4 TAATTTCTACTGTTGTAGATCTGATGGTCCAT (SEQ ID NO: 179) GTCTGTTACTC (SEQ ID NO: 180) Lb3Cpf1DNMT1 target 1 TAATTTCTACTGTTGTAGAT TTTCCCTTCAGCT (SEQ ID NO: 181)AAAATAAAGG (SEQ ID NO: 182) Lb3Cpf1 DNMT1 target 2 TAATTTCTACTAAGTGTAGATCCTCACTCCTGC (SEQ ID NO: 183) TCGGTGAATTT (SEQ ID NO: 184) Lb3Cpf1DNMT1 target 3 TAATTTCTACTAAGTGTAGAT AGGAGTGTTCAG (SEQ ID NO: 185)TCTCCGTGAAC (SEQ ID NO: 186) Lb3Cpf1 DNMT1 target 4TAATTTCTACTAAGTGTAGAT CTGATGGTCCAT (SEQ ID NO: 187) GTCTGTTACTC(SEQ ID NO: 188) SpCas9 DNMT1 target 1 TAATTTCTACTAAGTGTAGATTTTCCCTTCAGCT (SEQ ID NO: 189) AAAATAAAGG (SEQ ID NO: 190) SpCas9DNMT1 target 2 na TCACTCCTGCTC GGTGAATT (SEQ ID NO: 191) SpCas9DNMT1 target 3 na AACCCTCTGGGG ACCGTTTG (SEQ ID NO: 192) SpCas9DNMT1 target 4 na AGTACGTTAATG TTTCCTGA (SEQ ID NO: 193)

TABLE 10 Ortholog specific direct repeats for crRNAstargeting proto-spacer 1 and DNMT1 target 3 Direct repeat originSequence FnCpf1 TAATTTCTACTGTTGTAGAT (SEQ ID NO: 195) Lb1Cpf1AGAAATGCATGGTTCTCATGC (SEQ ID NO: 196) BpCpf1AAAATTACCTAGTAATTAGGT (SEQ ID NO: 197) PeCpf1GGATTTCTACTTTTGTAGAT (SEQ ID NO: 198) PbCpf1AAATTTCTACTTTTGTAGAT (SEQ ID NO: 199) SsCpf1CGCGCCCACGCGGGGCGCGAC (SEQ ID NO: 200) AsCpf1TAATTTCTACTCTTGTAGAT (SEQ ID NO: 201) Lb2Cpf1GAATTTCTACTATTGTAGAT (SEQ ID NO: 202) CMtCpf1GAATCTCTACTCTTTGTAGAT (SEQ ID NO: 203) EeCpf1TAATTTCTACTTTGTAGAT (SEQ ID NO: 204) MbCpf1AAATTTCTACTGTTTGTAGAT (SEQ ID NO: 205) LiCpf1GAATTTCTACTTTTGTAGAT (SEQ ID NO: 206) Lb3Cpf1TAATTTCTACTAAGTGTAGAT (SEQ ID NO: 207) PcCpf1TAATTTCTACTATTGTAGAT (SEQ ID NO: 208) PdCpf1TAATTTCTACTTCGGTAGAT (SEQ ID NO: 209) PmCpf1TAATTTCTACTATTGTAGAT (SEQ ID NO: 210)

Example 8: Cloning of Francisella tularensis Subsp. Novicida U112 Cpf1(FnCpf1)

Applicants cloned the Francisella tularensis subsp. novicida U112 (FIG.95A) Cpf1 (FnCpf1) locus into low-copy plasmids (pFnCpf1) to allowheterologous reconstitution in Escherichia coli. Typically, in currentlycharacterized CRISPR-Cas systems, there are two requirements for DNAinterference: (i) the target sequence has to match one of the spacerspresent in the respective CRISPR array, and (ii) the target sequencecomplementary to the spacer (hereinafter protospacer) has to be flankedby the appropriate Protospacer-Adjacent Motif (PAM). Given thecompletely uncharacterized functionality of the FnCpf1 CRISPR locus, aplasmid depletion assay was designed to ascertain the activity of Cpf1and identify PAM sequence and its respective location relative to theprotospacer (5′ or 3′) (FIG. 95B). Two libraries of plasmids carrying aprotospacer matching the first spacer in the FnCpf1 CRISPR array wereconstructed with the 5′ or 3′ 7 bp sequences randomized. Each plasmidlibrary was transformed into E. coli that heterologously expressed theFnCpf1 locus or into a control E. coli strain carrying the empty vector.Using this assay, the PAM sequence and location was determined byidentifying nucleotide motifs that are preferentially depleted in cellsheterologously expressing the FnCpf1 locus. The PAM for FnCpf1 was foundto be located upstream of the 5′ end of displaced strand of theprotospacer and has the sequence 5′-TTN (FIGS. 95C-D and 102). The 5′location of the PAM is also observed in type I CRISPR systems, but notin type II systems, where Cas9 employs PAM sequences that are on the 3′end of the protospacer (Mojica et al., 2009; Garneau et al., 2010.Beyond the identification of the PAM, the results of the depletion assayclearly indicate that heterologously expressed Cpf1 loci are capable ofefficient interference with plasmid DNA.

To further characterize the PAM, plasmid interference activity wasanalyzed by transforming cpf1-locus expressing cells with plasmidscarrying protospacer 1 flanked by 5′-TTN PAMs. All 5′-TTN PAMs wereefficiently targeted (FIG. 1E). In addition, 5′-CTA but not 5′-TCA wasalso efficiently targeted (FIG. 95E), suggesting that the middle T ismore critical for PAM recognition than the first T and that, inagreement with the sequence motifs depleted in the PAM discovery assay(FIG. 102D), the PAM might be more relaxed than 5′-TTN.

Example 9: The Cpf1 CRISPR Array is Processed Independent of tracrRNA

Small RNAseq was used to determine the exact identity of the crRNAproduced by the cpf1-based CRISPR loci. By sequencing small RNAsextracted from a Francisella tularensis subsp. novicida U112 culture, itwas found that the CRISPR array is processed into short mature crRNAs of42-44 nt in length. Each mature crRNA begins with 19 nt of the directrepeat followed by 23-25 nt of the spacer sequence (FIG. 96A). ThiscrRNA arrangement contrasts with that in type II CRISPR-Cas systemswhere the mature crRNA begins with 20-24 nt of spacer sequence followedby ˜22 nt of direct repeat (Deltcheva et al., 2011; Chylinski et al.,2013). Unexpectedly, apart from the crRNAs, we did not observe anyrobustly expressed small transcripts near the Francisella cpf1 locusthat might correspond to tracrRNAs, which are associated with Cas9-basedsystems.

To confirm that no additional RNAs are required for crRNA maturation andDNA interference, an expression plasmid was constructed using syntheticpromoters to drive the expression of Francisella cpf1 (FnCpf1) and theCRISPR array (pFnCpf1 min). Small RNAseq of E. coli expressing thisplasmid still showed robust processing of the CRISPR array into maturecrRNA (FIG. 96B), indicating that FnCpf1 and its CRISPR array aresufficient to achieve crRNA processing. Furthermore, E. coli expressingpFnCpf1_min as well as pFnCpf1_ΔCas, a plasmid with all of the cas genesremoved but retaining native promoters driving the expression of FnCpf1and the CRISPR array, also exhibited robust DNA interference,demonstrating that FnCpf1 and crRNA are sufficient for mediating DNAtargeting (FIG. 96C). By contrast, Cas9 requires both crRNA and tracrRNAto mediate targeted DNA interference (Deltcheva et al., 2011; Zhang etal., 2013).

Example 10: Cpf1 is a Single crRNA Guided Endonuclease

The finding that FnCpf1 can mediate DNA interference with crRNA alone ishighly surprising given that Cas9 recognizes crRNA through the duplexstructure between crRNA and tracrRNA (Jinek et al., 2012; Nishimasu etal., 2014), as well as the 3′ secondary structure of the tracrRNA (Hsuet al., 2013; Nishimasu et al., 2014). To ensure that crRNA is indeedsufficient for forming an active complex with FnCpf1 and mediatingRNA-guided DNA cleavage, FnCpf1 supplied only with crRNA was tested fortarget DNA cleavage in vitro. Purified FnCpf1 (FIG. 103 ) was assayedfor its ability to cleave the same protospacer 1-containing plasmid usedin the bacterial DNA interference experiments (FIG. 97A). FnCpf1 with anin vitro transcribed mature crRNA targeting protospacer 1 was able toefficiently cleave the target plasmid in a Mg²⁺- and crRNA-dependentmanner (FIG. 97B). Moreover, FnCpf1 was able to cleave both supercoiledand linear target DNA (FIG. 97C). These results clearly demonstrate thesufficiency of FnCpf1 and crRNA for RNA-guided DNA cleavage.

The cleavage site of FnCpf1 was also mapped using Sanger sequencing ofthe cleaved DNA ends. FnCpf1-mediated cleavage results in a 5-nt 5′overhang (FIGS. 97A, 97D, and 104 ), which is distinct from the bluntcleavage product generated by Cas9 (Garneau et al., 2010; Jinek et al.,2012; Gasiunas et al., 2012). The staggered cleavage site of FnCpf1 isdistant from the PAM: cleavage occurs after the 18th base on thenon-targeted (+) strand and after the 23rd base on the targeted (−)strand (FIGS. 97A, 97D, and 104 ). Using double-stranded oligosubstrates with different PAM sequences, we also found that FnCpf1cleave the target DNA when the 5′-TTN PAM to be in a duplex form (FIG.97E), in contrast to the PAMs of Cas9 (Sternberg et al., 2014).

Example 11: The RuvC-Like Domain of Cpf1 Mediates RNA-Guided DNACleavage

The RuvC-like domain of Cpf1 retains all the catalytic residues of thisfamily of endonucleases (FIGS. 98A and 105 ) and is thus predicted to bean active nuclease. Three mutants, FnCpf1(D917A), FnCpf1(E1006A), andFnCpf1(D1225A) (FIG. 98A) were generated to test whether the conservedcatalytic residues are essential for the nuclease activity of FnCpf1.The D917A and E1006A mutations completely inactivated the DNA cleavageactivity of FnCpf1, and D1255A significantly reduced nucleolyticactivity (FIG. 98B). These results are in contrast to the mutagenesisresults for Streptococcus pyogenes Cas9 (SpCas9), where mutation of theRuvC (D10A) and HNH (N863A) nuclease domains converts SpCas9 into a DNAnickase (i.e. inactivation of each of the two nuclease domains abolishedthe cleavage of one of the DNA strands) (Jinek et al., 2012; Gasiunas etal., 2012) (FIG. 98B). These findings suggest that the RuvC-like domainof FnCpf1 cleaves both strands of the target DNA, perhaps in a dimericconfiguration (FIG. 103B).

Example 12: Sequence and Structure of the Cpf1 crRNA

Compared with the guide RNA for Cas9, which has elaborate RNA secondarystructure features that interact with Cas9 (Nishimasu et al., 2014), theguide RNA for FnCpf1 is notably simpler and only comprises a single stemloop in the direct repeat sequence (FIG. 97A).

The sequence and structural requirements of crRNA for mediating DNAcleavage with FnCpf1 were explored. The length of the guide sequence wasexamined. A 16 nt guide sequence was observed to achieve detectable DNAcleavage and guide sequences of 18 nt achieved efficient DNA cleavage invitro (FIG. 99A). These lengths are similar to those demonstrated forSpCas9 where a 16 to 17 nt spacer sequence is sufficient for DNAcleavage (Cencic et al., 2014; Fu et al., 2014). The seed region of theFnCpf1 guide RNA was observed within the first 6 or 7 nt on the 5′ endof the spacer sequence (FIG. 99B).

The effect of direct repeat mutations on the RNA-guided DNA cleavageactivity was investigated. The direct repeat portion of mature crRNA is19 nt long (FIG. 96A). Truncation of the direct repeat revealed that 16nt is sufficient, but optimally more than 17 nt of the direct repeat iseffective for cleavage. Mutations in the stem loop that preserved theRNA duplex did not affect the cleavage activity, whereas mutations thatdisrupted the stem loop duplex structure abolished cleavage (FIG. 99D).Finally, base substitutions in the loop region did not affect nucleaseactivity, whereas substitution of the U immediately 5′ of the spacersequence reduced activity substantially (FIG. 5E). Collectively, theseresults suggest that FnCpf1 recognizes the crRNA through a combinationof sequence-specific and structural features of the stem loop.

Example 13: Cpf1-Family Proteins from Diverse Bacteria Share CommoncrRNA Structures and PAMs

To investigate the use of Cpf1 as a genome editing tool, the diversityof Cpf1-family proteins available in the public sequences databases wasexploited. A BLAST search of the WGS database at the NCBI revealed 46non-redundant Cpf1-family proteins (FIG. 64 ). 16 were chosen based onour phylogenetic reconstruction (FIG. 64 ), as representative of Cpf1diversity (FIGS. 100A-100B and 106 ). These Cpf1-family proteins span arange of lengths between ˜1200 and ˜1500 amino acids.

The direct repeat sequences for each of these Cpf1-family proteins showstrong conservation in the 19 nucleotides at the 3′ of the directrepeat, the portion of the repeat that is included in the processedcrRNA (FIG. 100C). The 5′ sequence of the direct repeat is much morediverse. Of the 16 Cpf1-family proteins chosen for analysis, three(2—Lachnospiraceae bacterium MC2017, Lb3Cpf1; 3—Butyrivibrioproteoclasticus, BpCpf1; and 6—Smithella sp. SC_K08D17, SsCpf1) wereassociated with direct repeat sequences that are notably divergent fromthe FnCpf1 direct repeat (FIG. 100C). Notably, these direct repeatsequences preserved stem loop structures that were identical ornearly-identical to the FnCpf1 direct repeat (FIG. 100D).

Orthologous direct repeat sequences are tested for the ability tosupport FnCpf1 nuclease activity in vitro. Direct repeats that containedconserved stem sequences were able to function interchangeably withFnCpf1. The direct repeat from candidate 3 (BpCpf1) supported a lowlevel of FnCpf1 nuclease activity (FIG. 100E), possibly due to theconservation of the 3′-most U.

An in vitro PAM identification assay (FIG. 107A) was used to determinethe PAM sequence for each Cpf1-family protein. PAM sequences wereidentified for 7 new Cpf1-family proteins (FIGS. 100E and 107B-C), andthe screen confirmed the PAM for FnCpf1 as 5′-TTN. The PAM sequences forthe Cpf1-family proteins were predominantly T-rich, varying primarily inthe number of Ts constituting each PAM (FIGS. 100F and 107B-C).

Example 14: Cpf1 can be Harnessed to Facilitate Genome Editing in HumanCells

Cpf1-family proteins were codon optimized and attached a C-terminalnuclear localization signal (NLS) for optimal expression and nucleartargeting in human cells (FIG. 101A). To test the activity of eachCpf1-family protein, a guide RNA target site was selected within theDNMT1 gene (FIG. 101B). Each of the Cpf1-family proteins along with itsrespective crRNA designed to target DNMT1 was able to cleave a PCRamplicon of the DNMT1 genomic region in vitro (FIG. 101C). When testedin human embryonic kidney 293FT (HEK 293FT) cells, 2 of the Cpf1-familyproteins (7-AsCpf1 and 13-LbCpf1) exhibited detectable levels ofnuclease-induced indels under the conditions employed (FIGS. 101C andD).

Each Cpf1-family protein was tested with additional genomic targets.AsCpf1 and LbCpf1 consistently mediated robust genome editing inHEK293FT cells (FIGS. 101E and 108 ). When compared to Cas9, AsCpf1 andLbCpf1 mediated comparable levels of indel formation (FIG. 101E).Additionally, we used in vitro cleavage followed by Sanger sequencing ofthe cleaved DNA ends and found that 7—AsCpf1 and 13—LbCpf1 alsogenerated staggered cleavage sites (FIGS. 101D and 107E).

Following are nucleotide and amino acid sequences of FnCpf1 constructsand orthologs:

FnCpf1 locus sequences pFnCpf15'end of endogenous F. novicida acetyltranferase (upstream of FnCpfllocus) FnCpf1

Cas2 Direct repeats Spacer (SEQ ID NO: 211)CATCAAGGAATTGGTTCTAAGCTTATAGAAGCAATGATTAAGGAAGCCAAAAAAAATAATATTGATGCAATATTTGTCTTAGGTCATCCAAGTTATTATCCAAAATTTGGTTTTAAACCAGCCACAGAATATCAGATAAAATGTGAATATGATGTCCCAGCGGATGTTTTTATGGTACTAGATTTGTCAGCTAAACTAGCTAGTTTAAAAGGACAAACTGTCTACTATGCCGATGAGTTTGGCAAAATTTTTTAGATCTACAAAATTATAAACTAAATAAAGATTCTTATAATAACTTTATATATAATCGAAATGTAGAGAATTTTATAAGGAGTCTTTATCATGTCAATTTATCAAGAATTTGTTAATAAATATAGTTTAAGTAAAACTCTAAGATTTGAGTTAATCCCACAGGGTAAAACACTTGAAAACATAAAAGCAAGAGGTTTGATTTTAGATGATGAGAAAAGAGCTAAAGACTACAAAAAGGCTAAACAAATAATTGATAAATATCATCAGTTTTTTATAGAGGAGATATTAAGTTCGGTTTGTATTAGCGAAGATTTATTACAAAACTATTCTGATGTTTATTTTAAACTTAAAAAGAGTGATGATGATAATCTACAAAAAGATTTTAAAAGTGCAAAAGATACGATAAAGAAACAAATATCTGAATATATAAAGGACTCAGAGAAATTTAAGAATTTGTTTAATCAAAACCTTATCGATGCTAAAAAAGGGCAAGAGTCAGATTTAATTCTATGGCTAAAGCAATCTAAGGATAATGGTATAGAACTATTTAAAGCCAATAGTGATATCACAGATATAGATGAGGCGTTAGAAATAATCAAATCTTTTAAAGGTTGGACAACTTATTTTAAGGGTTTTCATGAAAATAGAAAAAATGTTTATAGTAGCAATGATATTCCTACATCTATTATTTATAGGATAGTAGATGATAATTTGCCTAAATTTCTAGAAAATAAAGCTAAGTATGAGAGTTTAAAAGACAAAGCTCCAGAAGCTATAAACTATGAACAAATTAAAAAAGATTTGGCAGAAGAGCTAACCTTTGATATTGACTACAAAACATCTGAAGTTAATCAAAGAGTTTTTTCACTTGATGAAGTTTTTGAGATAGCAAACTTTAATAATTATCTAAATCAAAGTGGTATTACTAAATTTAATACTATTATTGGTGGTAAATTTGTAAATGGTGAAAATACAAAGAGAAAAGGTATAAATGAATATATAAATCTATACTCACAGCAAATAAATGATAAAACACTCAAAAAATATAAAATGAGTGTTTTATTTAAGCAAATTTTAAGTGATACAGAATCTAAATCTTTTGTAATTGATAAGTTAGAAGATGATAGTGATGTAGTTACAACGATGCAAAGTTTTTATGAGCAAATAGCAGCTTTTAAAACAGTAGAAGAAAAATCTATTAAAGAAACACTATCTTTATTATTTGATGATTTAAAAGCTCAAAAACTTGATTTGAGTAAAATTTATTTTAAAAATGATAAATCTCTTACTGATCTATCACAACAAGTTTTTGATGATTATAGTGTTATTGGTACAGCGGTACTAGAATATATAACTCAACAAATAGCACCTAAAAATCTTGATAACCCTAGTAAGAAAGAGCAAGAATTAATAGCCAAAAAAACTGAAAAAGCAAAATACTTATCTCTAGAAACTATAAAGCTTGCCTTAGAAGAATTTAATAAGCATAGAGATATAGATAAACAGTGTAGGTTTGAAGAAATACTTGCAAACTTTGCGGCTATTCCGATGATATTTGATGAAATAGCTCAAAACAAAGACAATTTGGCACAGATATCTATCAAATATCAAAATCAAGGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTATCAAGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATATTTCATATTAGTCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAGCATTTTTATCTAGTATTTGAGGAGTGCTACTTTGAGCTAGCGAATATAGTGCCTCTTTATAACAAAATTAGAAACTATATAACTCAAAAGCCATATAGTGATGAGAAATTTAAGCTCAATTTTGAGAACTCGACTTTGGCTAATGGTTGGGATAAAAATAAAGAGCCTGACAATACGGCAATTTTATTTATCAAAGATGATAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATATTTGATGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTATAAACTTTTACCTGGCGCAAATAAAATGTTACCTAAGGTTTTCTTTTCTGCTAAATCTATAAAATTTTATAATCCTAGTGAAGATATACTTAGAATAAGAAATCATTCCACACATACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTTTAATATTGAAGATTGCCGAAAATTTATAGATTTTTATAAACAGTCTATAAGTAAGCATCCGGAGTGGAAAGATTTTGGATTTAGATTTTCTGATACTCAAAGATATAATTCTATAGATGAATTTTATAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATATCAGAGAGCTATATTGATAGCGTAGTTAATCAGGGTAAATTGTACCTATTCCAAATCTATAATAAAGATTTTTCAGCTTATAGCAAAGGGCGACCAAATCTACATACTTTATATTGGAAAGCGCTGTTTGATGAGAGAAATCTTCAAGATGTGGTTTATAAGCTAAATGGTGAGGCAGAGCTTTTTTATCGTAAACAATCAATACCTAAAAAAATCACTCACCCAGCTAAAGAGGCAATAGCTAATAAAAACAAAGATAATCCTAAAAAAGAGAGTGTTTTTGAATATGATTTAATCAAAGATAAACGCTTTACTGAAGATAAGTTTTTCTTTCACTGTCCTATTACAATCAATTTTAAATCTAGTGGAGCTAATAAGTTTAATGATGAAATCAATTTATTGCTAAAAGAAAAAGCAAATGATGTTCATATATTAAGTATAGATAGAGGTGAAAGACATTTAGCTTACTATACTTTGGTAGATGGTAAAGGCAATATCATCAAACAAGATACTTTCAACATCATTGGTAATGATAGAATGAAAACAAACTACCATGATAAGCTTGCTGCAATAGAGAAAGATAGGGATTCAGCTAGGAAAGACTGGAAAAAGATAAATAACATCAAAGAGATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAAATAGCTAAGCTAGTTATAGAGTATAATGCTATTGTGGTTTTTGAGGATTTAAATTTTGGATTTAAAAGAGGGCGTTTCAAGGTAGAGAAGCAGGTCTATCAAAAGTTAGAAAAAATGCTAATTGAGAAACTAAACTATCTAGTTTTCAAAGATAATGAGTTTGATAAAACTGGGGGAGTGCTTAGAGCTTATCAGCTAACAGCACCTTTTGAGACTTTTAAAAAGATGGGTAAACAAACAGGTATTATCTACTATGTACCAGCTGGTTTTACTTCAAAAATTTGTCCTGTAACTGGTTTTGTAAATCAGTTATATCCTAAGTATGAAAGTGTCAGCAAATCTCAAGAGTTCTTTAGTAAGTTTGACAAGATTTGTTATAACCTTGATAAGGGCTATTTTGAGTTTAGTTTTGATTATAAAAACTTTGGTGACAAGGCTGCCAAAGGCAAGTGGACTATAGCTAGCTTTGGGAGTAGATTGATTAACTTTAGAAATTCAGATAAAAATCATAATTGGGATACTCGAGAAGTTTATCCAACTAAAGAGTTGGAGAAATTGCTAAAAGATTATTCTATCGAATATGGGCATGGCGAATGTATCAAAGCAGCTATTTGCGGTGAGAGCGACAAAAAGTTTTTTGCTAAGCTAACTAGTGTCCTAAATACTATCTTACAAATGCGTAACTCAAAAACAGGTACTGAGTTAGATTATCTAATTTCACCAGTAGCAGATGTAAATGGCAATTTCTTTGATTCGCGACAGGCGCCAAAAAATATGCCTCAAGATGCTGATGCCAATGGTGCTTATCATATTGGGCTAAAAGGTCTGATGCTACTAGGTAGGATCAAAAATAATCAAGAGGGCAAAAAACTCAATTTGGTTATCAAAAATGAAGAGTATTTTGAGTTCGTGCAGAATAGGAATAACTAATTCATTCAAGAATATATTACCCTGTCAGTTTAGCGACTATTACCTCTTTAATAATTTGCAGGGGAATTATTTTAGTAATAGTAATATACACAAGAGTTATTGATTATATGGAAAATTATATTTAGATAACATGGTTAAATGATTT

TTTTTGATGGAGTGAAACTTAGTCTATCATTGGGGAATATAGTTATAAAAGATAAAGAAACTGATGAGGTGAAAACTAAGCTTTCTGTTCATAAAGTTCTTGCATTGTTTATCGTAGGTAATATGACGATGACCTCGCAACTTTTAGAGACCTGTAAGAAAAATGCTATACAGCTAGTTTTTATGAAAAATAGCTTTAGACCATATCTATGTTTTGGTGATATTGCTGAGGCTAATTTTTTAGCTAGATATAAGCAATATAGTGTAGTTGAGCAAGATATAAGTTTAGCAAGGATTTTTATAACATCAAAGATACGCAATCAACATAACTTAGTCAAAAGCCTAAGAGATAAAACTCCAGAGCAGCAAGAGATAGTCAAAAAGAATAAACAGCTAATAGCAGAGTTAGAAAATACAACAAGCCTAGCGGAGCTAATGGGTATAGAGGGCAATGTTGCCAAAAATTTCTTCAAAGGATTCTATGGACATTTAGATAGTTGGCAAGGGCGCAAACCTAGAATAAAACAGGATCCATATAATGTTGTTTTAGACTTGGGCTATAGTATGTTGTTTAATTTTGTAGAGTGTTTTTTGCGACTTTTTGGCTTTGATTTATACAAGGGCTTTTGTCATCAGACTTGGTATAAGCGTAAATCCCTAGTTTGTGACTTTGTTGAGCCATTTAGATGTATAGTGGATAACCAAGTTAGAAAATCATGGAATCTCGGGCAATTTTCTGTAGAGGATTTTGGTTGCAAAAATGAGCAGTTTTATATAAAAAAAGATAAAACAAAAGACTACTCAAAAATACTTTTTGCCGAGATTATCAGCTACAAGCTAGAGATATTTGAATATGTAAGAGAATTTTATCGTGCCTTTATGCGAGGCAAAGAAATTGCAGAGTATCCAATATTTTGTTATGAAACTAGGAGGGTGTATGTTGATAGTCAGTTATGATTTTAGTAATAATAAAGTACGTGCAAAGTTTGCCAAATTTCTAGAAAGTTATGGTGTACGTTTACAATATTCGGTATTTGAGCTCAAATATAGCAAGAGAATGTTAGACTTGATTTTAGCTGAGATAGAAAATAACTATGTACCACTATTTACAAATGCTGATAGTGTTTTAATCTTTAATGCTCCAGATAAAGATGTGATAAAATATGGTTATGCGATTCATAGAGAACAAGAGGTTGTTTTTATAGACTAAAAATTGCAAACCTTAGTCTTTATGTTAAAATAACTACTAAGTTCTTAGAGATATTTAAAAATATGACTGTTGTTATATATCAAAATGCTAAAAAAATCATAGATTTTAGGTCTTTTTTTGCTGATTTAGGCAAAAACGGGTCTAAGAACTTTAAATAATTTCTACTGTTGTAGAT GAGAAGTCATTTAATAAGGCCACTGTTAAAA GTCTAAGAACTTTAAATAATTTCTACTGTTGTAGAT GCTACTATTCCTGTGCCTTCAGATAATTCAGTCTAAGAACTTTAAATAATTTCTACTGTTGTAGATGTCTAGAGCCTTTTGTATTAGTAGCCGGTCTAAGAACTTTAAATAATTTCTACTGTTGTAGAT TAGCGATTTATGAAGGTCATTTTTTTGTCT pFnCpf1_min

Shine-Dalgarno sequence FnCpfl

Direct repeats Spacer (SEQ ID NO: 212)

AAGAATTTGTTAATAAATATAGTTTAAGTAAAACTCTAAGATTTGAGTTAATCCCACAGGGTAAAACACTTGAAAACATAAAAGCAAGAGGTTTGATTTTAGATGATGAGAAAAGAGCTAAAGACTACAAAAAGGCTAAACAAATAATTGATAAATATCATCAGTTTTTTATAGAGGAGATATTAAGTTCGGTTTGTATTAGCGAAGATTTATTACAAAACTATTCTGATGTTTATTTTAAACTTAAAAAGAGTGATGATGATAATCTACAAAAAGATTTTAAAAGTGCAAAAGATACGATAAAGAAACAAATATCTGAATATATAAAGGACTCAGAGAAATTTAAGAATTTGTTTAATCAAAACCTTATCGATGCTAAAAAAGGGCAAGAGTCAGATTTAATTCTATGGCTAAAGCAATCTAAGGATAATGGTATAGAACTATTTAAAGCCAATAGTGATATCACAGATATAGATGAGGCGTTAGAAATAATCAAATCTTTTAAAGGTTGGACAACTTATTTTAAGGGTTTTCATGAAAATAGAAAAAATGTTTATAGTAGCAATGATATTCCTACATCTATTATTTATAGGATAGTAGATGATAATTTGCCTAAATTTCTAGAAAATAAAGCTAAGTATGAGAGTTTAAAAGACAAAGCTCCAGAAGCTATAAACTATGAACAAATTAAAAAAGATTTGGCAGAAGAGCTAACCTTTGATATTGACTACAAAACATCTGAAGTTAATCAAAGAGTTTTTTCACTTGATGAAGTTTTTGAGATAGCAAACTTTAATAATTATCTAAATCAAAGTGGTATTACTAAATTTAATACTATTATTGGTGGTAAATTTGTAAATGGTGAAAATACAAAGAGAAAAGGTATAAATGAATATATAAATCTATACTCACAGCAAATAAATGATAAAACACTCAAAAAATATAAAATGAGTGTTTTATTTAAGCAAATTTTAAGTGATACAGAATCTAAATCTTTTGTAATTGATAAGTTAGAAGATGATAGTGATGTAGTTACAACGATGCAAAGTTTTTATGAGCAAATAGCAGCTTTTAAAACAGTAGAAGAAAAATCTATTAAAGAAACACTATCTTTATTATTTGATGATTTAAAAGCTCAAAAACTTGATTTGAGTAAAATTTATTTTAAAAATGATAAATCTCTTACTGATCTATCACAACAAGTTTTTGATGATTATAGTGTTATTGGTACAGCGGTACTAGAATATATAACTCAACAAATAGCACCTAAAAATCTTGATAACCCTAGTAAGAAAGAGCAAGAATTAATAGCCAAAAAAACTGAAAAAGCAAAATACTTATCTCTAGAAACTATAAAGCTTGCCTTAGAAGAATTTAATAAGCATAGAGATATAGATAAACAGTGTAGGTTTGAAGAAATACTTGCAAACTTTGCGGCTATTCCGATGATATTTGATGAAATAGCTCAAAACAAAGACAATTTGGCACAGATATCTATCAAATATCAAAATCAAGGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTATCAAGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATATTTCATATTAGTCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAGCATTTTTATCTAGTATTTGAGGAGTGCTACTTTGAGCTAGCGAATATAGTGCCTCTTTATAACAAAATTAGAAACTATATAACTCAAAAGCCATATAGTGATGAGAAATTTAAGCTCAATTTTGAGAACTCGACTTTGGCTAATGGTTGGGATAAAAATAAAGAGCCTGACAATACGGCAATTTTATTTATCAAAGATGATAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATATTTGATGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTATAAACTTTTACCTGGCGCAAATAAAATGTTACCTAAGGTTTTCTTTTCTGCTAAATCTATAAAATTTTATAATCCTAGTGAAGATATACTTAGAATAAGAAATCATTCCACACATACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTTTAATATTGAAGATTGCCGAAAATTTATAGATTTTTATAAACAGTCTATAAGTAAGCATCCGGAGTGGAAAGATTTTGGATTTAGATTTTCTGATACTCAAAGATATAATTCTATAGATGAATTTTATAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATATCAGAGAGCTATATTGATAGCGTAGTTAATCAGGGTAAATTGTACCTATTCCAAATCTATAATAAAGATTTTTCAGCTTATAGCAAAGGGCGACCAAATCTACATACTTTATATTGGAAAGCGCTGTTTGATGAGAGAAATCTTCAAGATGTGGTTTATAAGCTAAATGGTGAGGCAGAGCTTTTTTATCGTAAACAATCAATACCTAAAAAAATCACTCACCCAGCTAAAGAGGCAATAGCTAATAAAAACAAAGATAATCCTAAAAAAGAGAGTGTTTTTGAATATGATTTAATCAAAGATAAACGCTTTACTGAAGATAAGTTTTTCTTTCACTGTCCTATTACAATCAATTTTAAATCTAGTGGAGCTAATAAGTTTAATGATGAAATCAATTTATTGCTAAAAGAAAAAGCAAATGATGTTCATATATTAAGTATAGATAGAGGTGAAAGACATTTAGCTTACTATACTTTGGTAGATGGTAAAGGCAATATCATCAAACAAGATACTTTCAACATCATTGGTAATGATAGAATGAAAACAAACTACCATGATAAGCTTGCTGCAATAGAGAAAGATAGGGATTCAGCTAGGAAAGACTGGAAAAAGATAAATAACATCAAAGAGATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAAATAGCTAAGCTAGTTATAGAGTATAATGCTATTGTGGTTTTTGAGGATTTAAATTTTGGATTTAAAAGAGGGCGTTTCAAGGTAGAGAAGCAGGTCTATCAAAAGTTAGAAAAAATGCTAATTGAGAAACTAAACTATCTAGTTTTCAAAGATAATGAGTTTGATAAAACTGGGGGAGTGCTTAGAGCTTATCAGCTAACAGCACCTTTTGAGACTTTTAAAAAGATGGGTAAACAAACAGGTATTATCTACTATGTACCAGCTGGTTTTACTTCAAAAATTTGTCCTGTAACTGGTTTTGTAAATCAGTTATATCCTAAGTATGAAAGTGTCAGCAAATCTCAAGAGTTCTTTAGTAAGTTTGACAAGATTTGTTATAACCTTGATAAGGGCTATTTTGAGTTTAGTTTTGATTATAAAAACTTTGGTGACAAGGCTGCCAAAGGCAAGTGGACTATAGCTAGCTTTGGGAGTAGATTGATTAACTTTAGAAATTCAGATAAAAATCATAATTGGGATACTCGAGAAGTTTATCCAACTAAAGAGTTGGAGAAATTGCTAAAAGATTATTCTATCGAATATGGGCATGGCGAATGTATCAAAGCAGCTATTTGCGGTGAGAGCGACAAAAAGTTTTTTGCTAAGCTAACTAGTGTCCTAAATACTATCTTACAAATGCGTAACTCAAAAACAGGTACTGAGTTAGATTATCTAATTTCACCAGTAGCAGATGTAAATGGCAATTTCTTTGATTCGCGACAGGCGCCAAAAAATATGCCTCAAGATGCTGATGCCAATGGTGCTTATCATATTGGGCTAAAAGGTCTGATGCTACTAGGTAGGATCAAAAATAATCAAGAGGGCAAAAAACTCAATT

ATAATTTCTACTGTTGTAGATGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTCTAAGAACTTTAAATAATTTCTACTGTTGTAGAT GCTACTATTCCTGTGCCTTCAGATAATTCA GTCTAAGAACTTTAAATAATTTCTACTGTTGTAGA pFnCpfl_ΔCas5'end of endogenous F. novicida acetyltranferase (upstream of FnCpfl locusFnCpfl Direct repeats Spacer (SEQ ID NO: 213)CTGTCTACTATGCCGATGAGTTTGGCAAAATTTTTTAGATCTACAAAATTATAAACTAAATAAAGATTCTTATAATAACTTTATATATAATCGAAATGTAGAGAATTTTATAAGGAGTCTTTATCATGTCAATTTATCAAGAATTTGTTAATAAATATAGTTTAAGTAAAACTCTAAGATTTGAGTTAATCCCACAGGGTAAAACACTTGAAAACATAAAAGCAAGAGGTTTGATTTTAGATGATGAGAAAAGAGCTAAAGACTACAAAAAGGCTAAACAAATAATTGATAAATATCATCAGTTTTTTATAGAGGAGATATTAAGTTCGGTTTGTATTAGCGAAGATTTATTACAAAACTATTCTGATGTTTATTTTAAACTTAAAAAGAGTGATGATGATAATCTACAAAAAGATTTTAAAAGTGCAAAAGATACGATAAAGAAACAAATATCTGAATATATAAAGGACTCAGAGAAATTTAAGAATTTGTTTAATCAAAACCTTATCGATGCTAAAAAAGGGCAAGAGTCAGATTTAATTCTATGGCTAAAGCAATCTAAGGATAATGGTATAGAACTATTTAAAGCCAATAGTGATATCACAGATATAGATGAGGCGTTAGAAATAATCAAATCTTTTAAAGGTTGGACAACTTATTTTAAGGGTTTTCATGAAAATAGAAAAAATGTTTATAGTAGCAATGATATTCCTACATCTATTATTTATAGGATAGTAGATGATAATTTGCCTAAATTTCTAGAAAATAAAGCTAAGTATGAGAGTTTAAAAGACAAAGCTCCAGAAGCTATAAACTATGAACAAATTAAAAAAGATTTGGCAGAAGAGCTAACCTTTGATATTGACTACAAAACATCTGAAGTTAATCAAAGAGTTTTTTCACTTGATGAAGTTTTTGAGATAGCAAACTTTAATAATTATCTAAATCAAAGTGGTATTACTAAATTTAATACTATTATTGGTGGTAAATTTGTAAATGGTGAAAATACAAAGAGAAAAGGTATAAATGAATATATAAATCTATACTCACAGCAAATAAATGATAAAACACTCAAAAAATATAAAATGAGTGTTTTATTTAAGCAAATTTTAAGTGATACAGAATCTAAATCTTTTGTAATTGATAAGTTAGAAGATGATAGTGATGTAGTTACAACGATGCAAAGTTTTTATGAGCAAATAGCAGCTTTTAAAACAGTAGAAGAAAAATCTATTAAAGAAACACTATCTTTATTATTTGATGATTTAAAAGCTCAAAAACTTGATTTGAGTAAAATTTATTTTAAAAATGATAAATCTCTTACTGATCTATCACAACAAGTTTTTGATGATTATAGTGTTATTGGTACAGCGGTACTAGAATATATAACTCAACAAATAGCACCTAAAAATCTTGATAACCCTAGTAAGAAAGAGCAAGAATTAATAGCCAAAAAAACTGAAAAAGCAAAATACTTATCTCTAGAAACTATAAAGCTTGCCTTAGAAGAATTTAATAAGCATAGAGATATAGATAAACAGTGTAGGTTTGAAGAAATACTTGCAAACTTTGCGGCTATTCCGATGATATTTGATGAAATAGCTCAAAACAAAGACAATTTGGCACAGATATCTATCAAATATCAAAATCAAGGTAAAAAAGACCTACTTCAAGCTAGTGCGGAAGATGATGTTAAAGCTATCAAGGATCTTTTAGATCAAACTAATAATCTCTTACATAAACTAAAAATATTTCATATTAGTCAGTCAGAAGATAAGGCAAATATTTTAGACAAGGATGAGCATTTTTATCTAGTATTTGAGGAGTGCTACTTTGAGCTAGCGAATATAGTGCCTCTTTATAACAAAATTAGAAACTATATAACTCAAAAGCCATATAGTGATGAGAAATTTAAGCTCAATTTTGAGAACTCGACTTTGGCTAATGGTTGGGATAAAAATAAAGAGCCTGACAATACGGCAATTTTATTTATCAAAGATGATAAATATTATCTGGGTGTGATGAATAAGAAAAATAACAAAATATTTGATGATAAAGCTATCAAAGAAAATAAAGGCGAGGGTTATAAAAAAATTGTTTATAAACTTTTACCTGGCGCAAATAAAATGTTACCTAAGGTTTTCTTTTCTGCTAAATCTATAAAATTTTATAATCCTAGTGAAGATATACTTAGAATAAGAAATCATTCCACACATACAAAAAATGGTAGTCCTCAAAAAGGATATGAAAAATTTGAGTTTAATATTGAAGATTGCCGAAAATTTATAGATTTTTATAAACAGTCTATAAGTAAGCATCCGGAGTGGAAAGATTTTGGATTTAGATTTTCTGATACTCAAAGATATAATTCTATAGATGAATTTTATAGAGAAGTTGAAAATCAAGGCTACAAACTAACTTTTGAAAATATATCAGAGAGCTATATTGATAGCGTAGTTAATCAGGGTAAATTGTACCTATTCCAAATCTATAATAAAGATTTTTCAGCTTATAGCAAAGGGCGACCAAATCTACATACTTTATATTGGAAAGCGCTGTTTGATGAGAGAAATCTTCAAGATGTGGTTTATAAGCTAAATGGTGAGGCAGAGCTTTTTTATCGTAAACAATCAATACCTAAAAAAATCACTCACCCAGCTAAAGAGGCAATAGCTAATAAAAACAAAGATAATCCTAAAAAAGAGAGTGTTTTTGAATATGATTTAATCAAAGATAAACGCTTTACTGAAGATAAGTTTTTCTTTCACTGTCCTATTACAATCAATTTTAAATCTAGTGGAGCTAATAAGTTTAATGATGAAATCAATTTATTGCTAAAAGAAAAAGCAAATGATGTTCATATATTAAGTATAGATAGAGGTGAAAGACATTTAGCTTACTATACTTTGGTAGATGGTAAAGGCAATATCATCAAACAAGATACTTTCAACATCATTGGTAATGATAGAATGAAAACAAACTACCATGATAAGCTTGCTGCAATAGAGAAAGATAGGGATTCAGCTAGGAAAGACTGGAAAAAGATAAATAACATCAAAGAGATGAAAGAGGGCTATCTATCTCAGGTAGTTCATGAAATAGCTAAGCTAGTTATAGAGTATAATGCTATTGTGGTTTTTGAGGATTTAAATTTTGGATTTAAAAGAGGGCGTTTCAAGGTAGAGAAGCAGGTCTATCAAAAGTTAGAAAAAATGCTAATTGAGAAACTAAACTATCTAGTTTTCAAAGATAATGAGTTTGATAAAACTGGGGGAGTGCTTAGAGCTTATCAGCTAACAGCACCTTTTGAGACTTTTAAAAAGATGGGTAAACAAACAGGTATTATCTACTATGTACCAGCTGGTTTTACTTCAAAAATTTGTCCTGTAACTGGTTTTGTAAATCAGTTATATCCTAAGTATGAAAGTGTCAGCAAATCTCAAGAGTTCTTTAGTAAGTTTGACAAGATTTGTTATAACCTTGATAAGGGCTATTTTGAGTTTAGTTTTGATTATAAAAACTTTGGTGACAAGGCTGCCAAAGGCAAGTGGACTATAGCTAGCTTTGGGAGTAGATTGATTAACTTTAGAAATTCAGATAAAAATCATAATTGGGATACTCGAGAAGTTTATCCAACTAAAGAGTTGGAGAAATTGCTAAAAGATTATTCTATCGAATATGGGCATGGCGAATGTATCAAAGCAGCTATTTGCGGTGAGAGCGACAAAAAGTTTTTTGCTAAGCTAACTAGTGTCCTAAATACTATCTTACAAATGCGTAACTCAAAAACAGGTACTGAGTTAGATTATCTAATTTCACCAGTAGCAGATGTAAATGGCAATTTCTTTGATTCGCGACAGGCGCCAAAAAATATGCCTCAAGATGCTGATGCCAATGGTGCTTATCATATTGGGCTAAAAGGTCTGATGCTACTAGGTAGGATCAAAAATAATCAAGAGGGCAAAAAACTCAATTTGGTTATCAAAAATGAAGAGTATTTTGAGTTCGTGCAGAATAGGAATAACTAATTCATTCAAGAATATATTACCCTGTCAGTTTAGCGACTATTACCTCTTTAATAATTTGCAGGGGAATTATTTTAGTAATAGTAATATACACAAGAGTTATTGATTATATGGAAAATTATATTTAGATAACATGGTTAAATGATTTTATATTCTGTCCTTACTCGATATATTTTTTATAGACTAAAAATTGCAAACCTTAGTCTTTATGTTAAAATAACTACTAAGTTCTTAGAGATATTTAAAAATATGACTGTTGTTATATATCAAAATGCTAAAAAAATCATAGATTTTAGGTCTTTTTTTGCTGATTTAGGCAAAAACGGGTCTAAGAACTTTAAATAATTTCTACTGTTGTAGAT GAGAAGTCATTTAATAAGGCCACTGTTAAAA GTCTAAGAACTTTAAATAATTTCTACTGTTGTAGAT GCTACTATTCCTGTGCCTTCAGATAATTCA GTCTAAGAACTTTAAATAATTTCTACTGTTGT AGATGTCTAGAGCCTTTTGTATTAGTAGCCG GTCTAAGAACTTTAAATAATTTCTACTGTT GTAGATTAGCGATTTATGAAGGTCATTTTTTTGTCTNucleotide sSequences of human codon optimized Cpfl orthologsNuclear localization signal (NLS) Glycine-Serine linker 3x HA tag1- Francisella tularensis subsp. Novicida U112 (FnCpfl) (SEQ ID NO: 214)ATGAGCATCTACCAGGAGTTCGTCAACAAGTATTCACTGAGTAAGACACTGCGGTTCGAGCTGATCCCACAGGGCAAGACACTGGAGAACATCAAGGCCCGAGGCCTGATTCTGGACGATGAGAAGCGGGCAAAAGACTATAAGAAAGCCAAGCAGATCATTGATAAATACCACCAGTTCTTTATCGAGGAAATTCTGAGCTCCGTGTGCATCAGTGAGGATCTGCTGCAGAATTACTCAGACGTGTACTTCAAGCTGAAGAAGAGCGACGATGACAACCTGCAGAAGGACTTCAAGTCCGCCAAGGACACCATCAAGAAACAGATTAGCGAGTACATCAAGGACTCCGAAAAGTTTAAAAATCTGTTCAACCAGAATCTGATCGATGCTAAGAAAGGCCAGGAGTCCGACCTGATCCTGTGGCTGAAACAGTCTAAGGACAATGGGATTGAACTGTTCAAGGCTAACTCCGATATCACTGATATTGACGAGGCACTGGAAATCATCAAGAGCTTCAAGGGATGGACCACATACTTTAAAGGCTTCCACGAGAACCGCAAGAACGTGTACTCCAGCAACGACATTCCTACCTCCATCATCTACCGAATCGTCGATGACAATCTGCCAAAGTTCCTGGAGAACAAGGCCAAATATGAATCTCTGAAGGACAAAGCTCCCGAGGCAATTAATTACGAACAGATCAAGAAAGATCTGGCTGAGGAACTGACATTCGATATCGACTATAAGACTAGCGAGGTGAACCAGAGGGTCTTTTCCCTGGACGAGGTGTTTGAAATCGCCAATTTCAACAATTACCTGAACCAGTCCGGCATTACTAAATTCAATACCATCATTGGCGGGAAGTTTGTGAACGGGGAGAATACCAAGCGCAAGGGAATTAACGAATACATCAATCTGTATAGCCAGCAGATCAACGACAAAACTCTGAAGAAATACAAGATGTCTGTGCTGTTCAAACAGATCCTGAGTGATACCGAGTCCAAGTCTTTTGTCATTGATAAACTGGAAGATGACTCAGACGTGGTCACTACCATGCAGAGCTTTTATGAGCAGATCGCCGCTTTCAAGACAGTGGAGGAAAAATCTATTAAGGAAACTCTGAGTCTGCTGTTCGATGACCTGAAAGCCCAGAAGCTGGACCTGAGTAAGATCTACTTCAAAAACGATAAGAGTCTGACAGACCTGTCACAGCAGGTGTTTGATGACTATTCCGTGATTGGGACCGCCGTCCTGGAGTACATTACACAGCAGATCGCTCCAAAGAACCTGGATAATCCCTCTAAGAAAGAGCAGGAACTGATCGCTAAGAAAACCGAGAAGGCAAAATATCTGAGTCTGGAAACAATTAAGCTGGCACTGGAGGAGTTCAACAAGCACAGGGATATTGACAAACAGTGCCGCTTTGAGGAAATCCTGGCCAACTTCGCAGCCATCCCCATGATTTTTGATGAGATCGCCCAGAACAAAGACAATCTGGCTCAGATCAGTATTAAGTACCAGAACCAGGGCAAGAAAGACCTGCTGCAGGCTTCAGCAGAAGATGACGTGAAAGCCATCAAGGATCTGCTGGACCAGACCAACAATCTGCTGCACAAGCTGAAAATCTTCCATATTAGTCAGTCAGAGGATAAGGCTAATATCCTGGATAAAGACGAACACTTCTACCTGGTGTTCGAGGAATGTTACTTCGAGCTGGCAAACATTGTCCCCCTGTATAACAAGATTAGGAACTACATCACACAGAAGCCTTACTCTGACGAGAAGTTTAAACTGAACTTCGAAAATAGTACCCTGGCCAACGGGTGGGATAAGAACAAGGAGCCTGACAACACAGCTATCCTGTTCATCAAGGATGACAAGTACTATCTGGGAGTGATGAATAAGAAAAACAATAAGATCTTCGATGACAAAGCCATTAAGGAGAACAAAGGGGAAGGATACAAGAAAATCGTGTATAAGCTGCTGCCCGGCGCAAATAAGATGCTGCCTAAGGTGTTCTTCAGCGCCAAGAGTATCAAATTCTACAACCCATCCGAGGACATCCTGCGGATTAGAAATCACTCAACACATACTAAGAACGGGAGCCCCCAGAAGGGATATGAGAAATTTGAGTTCAACATCGAGGATTGCAGGAAGTTTATTGACTTCTACAAGCAGAGCATCTCCAAACACCCTGAATGGAAGGATTTTGGCTTCCGGTTTTCCGACACACAGAGATATAACTCTATCGACGAGTTCTACCGCGAGGTGGAAAATCAGGGGTATAAGCTGACTTTTGAGAACATTTCTGAAAGTTACATCGACAGCGTGGTCAATCAGGGAAAGCTGTACCTGTTCCAGATCTATAACAAAGATTTTTCAGCATACAGCAAGGGCAGACCAAACCTGCATACACTGTACTGGAAGGCCCTGTTCGATGAGAGGAATCTGCAGGACGTGGTCTATAAACTGAACGGAGAGGCCGAACTGTTTTACCGGAAGCAGTCTATTCCTAAGAAAATCACTCACCCAGCTAAGGAGGCCATCGCTAACAAGAACAAGGACAATCCTAAGAAAGAGAGCGTGTTCGAATACGATCTGATTAAGGACAAGCGGTTCACCGAAGATAAGTTCTTTTTCCATTGTCCAATCACCATTAACTTCAAGTCAAGCGGCGCTAACAAGTTCAACGACGAGATCAATCTGCTGCTGAAGGAAAAAGCAAACGATGTGCACATCCTGAGCATTGACCGAGGAGAGCGGCATCTGGCCTACTATACCCTGGTGGATGGCAAAGGGAATATCATTAAGCAGGATACATTCAACATCATTGGCAATGACCGGATGAAAACCAACTACCACGATAAACTGGCTGCAATCGAGAAGGATAGAGACTCAGCTAGGAAGGACTGGAAGAAAATCAACAACATTAAGGAGATGAAGGAAGGCTATCTGAGCCAGGTGGTCCATGAGATTGCAAAGCTGGTCATCGAATACAATGCCATTGTGGTGTTCGAGGATCTGAACTTCGGCTTTAAGAGGGGGCGCTTTAAGGTGGAAAAACAGGTCTATCAGAAGCTGGAGAAAATGCTGATCGAAAAGCTGAATTACCTGGTGTTTAAAGATAACGAGTTCGACAAGACCGGAGGCGTCCTGAGAGCCTACCAGCTGACAGCTCCCTTTGAAACTTTCAAGAAAATGGGAAAACAGACAGGCATCATCTACTATGTGCCAGCCGGATTCACTTCCAAGATCTGCCCCGTGACCGGCTTTGTCAACCAGCTGTACCCTAAATATGAGTCAGTGAGCAAGTCCCAGGAATTTTTCAGCAAGTTCGATAAGATCTGTTATAATCTGGACAAGGGGTACTTCGAGTTTTCCTTCGATTACAAGAACTTCGGCGACAAGGCCGCTAAGGGGAAATGGACCATTGCCTCCTTCGGATCTCGCCTGATCAACTTTCGAAATTCCGATAAAAACCACAATTGGGACACTAGGGAGGTGTACCCAACCAAGGAGCTGGAAAAGCTGCTGAAAGACTACTCTATCGAGTATGGACATGGCGAATGCATCAAGGCAGCCATCTGTGGCGAGAGTGATAAGAAATTTTTCGCCAAGCTGACCTCAGTGCTGAATACAATCCTGCAGATGCGGAACTCAAAGACCGGGACAGAACTGGACTATCTGATTAGCCCCGTGGCTGATGTCAACGGAAACTTCTTCGACAGCAGACAGGCACCCAAAAATATGCCTCAGGATGCAGACGCCAACGGGGCCTACCACATCGGGCTGAAGGGACTGATGCTGCTGGGCCGGATCAAGAACAATCAGGAGGGGAAGAAGCTGAACCTGGTCATTAAGAACGAGGAATACTTCGAGTTTGTCCAGAATAGAAATAACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC3- Lachnospiraceae bacterium MC2017 (Lb3Cpf1) (SEQ ID NO: 215)ATGGATTACGGCAACGGCCAGTTTGAGCGGAGAGCCCCCCTGACCAAGACAATCACCCTGCGCCTGAAGCCTATCGGCGAGACACGGGAGACAATCCGCGAGCAGAAGCTGCTGGAGCAGGACGCCGCCTTCAGAAAGCTGGTGGAGACAGTGACCCCTATCGTGGACGATTGTATCAGGAAGATCGCCGATAACGCCCTGTGCCACTTTGGCACCGAGTATGACTTCAGCTGTCTGGGCAACGCCATCTCTAAGAATGACAGCAAGGCCATCAAGAAGGAGACAGAGAAGGTGGAGAAGCTGCTGGCCAAGGTGCTGACCGAGAATCTGCCAGATGGCCTGCGCAAGGTGAACGACATCAATTCCGCCGCCTTTATCCAGGATACACTGACCTCTTTCGTGCAGGACGATGCCGACAAGCGGGTGCTGATCCAGGAGCTGAAGGGCAAGACCGTGCTGATGCAGCGGTTCCTGACCACACGGATCACAGCCCTGACCGTGTGGCTGCCCGACAGAGTGTTCGAGAACTTTAATATCTTCATCGAGAACGCCGAGAAGATGAGAATCCTGCTGGACTCCCCTCTGAATGAGAAGATCATGAAGTTTGACCCAGATGCCGAGCAGTACGCCTCTCTGGAGTTCTATGGCCAGTGCCTGTCTCAGAAGGACATCGATAGCTACAACCTGATCATCTCCGGCATCTATGCCGACGATGAGGTGAAGAACCCTGGCATCAATGAGATCGTGAAGGAGTACAATCAGCAGATCCGGGGCGACAAGGATGAGTCCCCACTGCCCAAGCTGAAGAAGCTGCACAAGCAGATCCTGATGCCAGTGGAGAAGGCCTTCTTTGTGCGCGTGCTGTCTAACGACAGCGATGCCCGGAGCATCCTGGAGAAGATCCTGAAGGACACAGAGATGCTGCCCTCCAAGATCATCGAGGCCATGAAGGAGGCAGATGCAGGCGACATCGCCGTGTACGGCAGCCGGCTGCACGAGCTGAGCCACGTGATCTACGGCGATCACGGCAAGCTGTCCCAGATCATCTATGACAAGGAGTCCAAGAGGATCTCTGAGCTGATGGAGACACTGTCTCCAAAGGAGCGCAAGGAGAGCAAGAAGCGGCTGGAGGGCCTGGAGGAGCACATCAGAAAGTCTACATACACCTTCGACGAGCTGAACAGGTATGCCGAGAAGAATGTGATGGCAGCATACATCGCAGCAGTGGAGGAGTCTTGTGCCGAGATCATGAGAAAGGAGAAGGATCTGAGGACCCTGCTGAGCAAGGAGGACGTGAAGATCCGGGGCAACAGACACAATACACTGATCGTGAAGAACTACTTTAATGCCTGGACCGTGTTCCGGAACCTGATCAGAATCCTGAGGCGCAAGTCCGAGGCCGAGATCGACTCTGACTTCTACGATGTGCTGGACGATTCCGTGGAGGTGCTGTCTCTGACATACAAGGGCGAGAATCTGTGCCGCAGCTATATCACCAAGAAGATCGGCTCCGACCTGAAGCCCGAGATCGCCACATACGGCAGCGCCCTGAGGCCTAACAGCCGCTGGTGGTCCCCAGGAGAGAAGTTTAATGTGAAGTTCCACACCATCGTGCGGAGAGATGGCCGGCTGTACTATTTCATCCTGCCCAAGGGCGCCAAGCCTGTGGAGCTGGAGGACATGGATGGCGACATCGAGTGTCTGCAGATGAGAAAGATCCCTAACCCAACAATCTTTCTGCCCAAGCTGGTGTTCAAGGACCCTGAGGCCTTCTTTAGGGATAATCCAGAGGCCGACGAGTTCGTGTTTCTGAGCGGCATGAAGGCCCCCGTGACAATCACCAGAGAGACATACGAGGCCTACAGGTATAAGCTGTATACCGTGGGCAAGCTGCGCGATGGCGAGGTGTCCGAAGAGGAGTACAAGCGGGCCCTGCTGCAGGTGCTGACCGCCTACAAGGAGTTTCTGGAGAACAGAATGATCTATGCCGACCTGAATTTCGGCTTTAAGGATCTGGAGGAGTATAAGGACAGCTCCGAGTTTATCAAGCAGGTGGAGACACACAACACCTTCATGTGCTGGGCCAAGGTGTCTAGCTCCCAGCTGGACGATCTGGTGAAGTCTGGCAACGGCCTGCTGTTCGAGATCTGGAGCGAGCGCCTGGAGTCCTACTATAAGTACGGCAATGAGAAGGTGCTGCGGGGCTATGAGGGCGTGCTGCTGAGCATCCTGAAGGATGAGAACCTGGTGTCCATGCGGACCCTGCTGAACAGCCGGCCCATGCTGGTGTACCGGCCAAAGGAGTCTAGCAAGCCTATGGTGGTGCACCGGGATGGCAGCAGAGTGGTGGACAGGTTTGATAAGGACGGCAAGTACATCCCCCCTGAGGTGCACGACGAGCTGTATCGCTTCTTTAACAATCTGCTGATCAAGGAGAAGCTGGGCGAGAAGGCCCGGAAGATCCTGGACAACAAGAAGGTGAAGGTGAAGGTGCTGGAGAGCGAGAGAGTGAAGTGGTCCAAGTTCTACGATGAGCAGTTTGCCGTGACCTTCAGCGTGAAGAAGAACGCCGATTGTCTGGACACCACAAAGGACCTGAATGCCGAAGTGATGGAGCAGTATAGCGAGTCCAACAGACTGATCCTGATCAGGAATACCACAGATATCCTGTACTATCTGGTGCTGGACAAGAATGGCAAGGTGCTGAAGCAGAGATCCCTGAACATCATCAATGACGGCGCCAGGGATGTGGACTGGAAGGAGAGGTTCCGCCAGGTGACAAAGGATAGAAACGAGGGCTACAATGAGTGGGATTATTCCAGGACCTCTAACGACCTGAAGGAGGTGTACCTGAATTATGCCCTGAAGGAGATCGCCGAGGCCGTGATCGAGTACAACGCCATCCTGATCATCGAGAAGATGTCTAATGCCTTTAAGGACAAGTATAGCTTCCTGGACGACGTGACCTTCAAGGGCTTCGAGACAAAGCTGCTGGCCAAGCTGAGCGATCTGCACTTTAGGGGCATCAAGGACGGCGAGCCATGTTCCTTCACAAACCCCCTGCAGCTGTGCCAGAACGATTCTAATAAGATCCTGCAGGACGGCGTGATCTTTATGGTGCCAAATTCTATGACACGGAGCCTGGACCCCGACACCGGCTTCATCTTTGCCATCAACGACCACAATATCAGGACCAAGAAGGCCAAGCTGAACTTTCTGAGCAAGTTCGATCAGCTGAAGGTGTCCTCTGAGGGCTGCCTGATCATGAAGTACAGCGGCGATTCCCTGCCTACACACAACACCGACAATCGCGTGTGGAACTGCTGTTGCAATCACCCAATCACAAACTATGACCGGGAGACAAAGAAGGTGGAGTTCATCGAGGAGCCCGTGGAGGAGCTGTCCCGCGTGCTGGAGGAGAATGGCATCGAGACAGACACCGAGCTGAACAAGCTGAATGAGCGGGAGAACGTGCCTGGCAAGGTGGTGGATGCCATCTACTCTCTGGTGCTGAATTATCTGCGCGGCACAGTGAGCGGAGTGGCAGGACAGAGGGCCGTGTACTATAGCCCTGTGACCGGCAAGAAGTACGATATCTCCTTTATCCAGGCCATGAACCTGAATAGGAAGTGTGACTACTATAGGATCGGCTCCAAGGAGAGGGGAGAGTGGACCGATTTCGTGGCCCAGCTGATCAACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC4- Butyrivibrio proteoclasticus (BpCpfl) (SEQ ID NO: 216)ATGAGCATCTACCAGGAGTTCGTCAACAAGTATTCACTGAGTAAGACACTGCGGTTCGAGCTGATCCCACAGGGCAAGACACTGGAGAACATCAAGGCCCGAGGCCTGATTCTGGACGATGAGAAGCGGGCAAAAGACTATAAGAAAGCCAAGCAGATCATTGATAAATACCACCAGTTCTTTATCGAGGAAATTCTGAGCTCCGTGTGCATCAGTGAGGATCTGCTGCAGAATTACTCAGACGTGTACTTCAAGCTGAAGAAGAGCGACGATGACAACCTGCAGAAGGACTTCAAGTCCGCCAAGGACACCATCAAGAAACAGATTAGCGAGTACATCAAGGACTCCGAAAAGTTTAAAAATCTGTTCAACCAGAATCTGATCGATGCTAAGAAAGGCCAGGAGTCCGACCTGATCCTGTGGCTGAAACAGTCTAAGGACAATGGGATTGAACTGTTCAAGGCTAACTCCGATATCACTGATATTGACGAGGCACTGGAAATCATCAAGAGCTTCAAGGGATGGACCACATACTTTAAAGGCTTCCACGAGAACCGCAAGAACGTGTACTCCAGCAACGACATTCCTACCTCCATCATCTACCGAATCGTCGATGACAATCTGCCAAAGTTCCTGGAGAACAAGGCCAAATATGAATCTCTGAAGGACAAAGCTCCCGAGGCAATTAATTACGAACAGATCAAGAAAGATCTGGCTGAGGAACTGACATTCGATATCGACTATAAGACTAGCGAGGTGAACCAGAGGGTCTTTTCCCTGGACGAGGTGTTTGAAATCGCCAATTTCAACAATTACCTGAACCAGTCCGGCATTACTAAATTCAATACCATCATTGGCGGGAAGTTTGTGAACGGGGAGAATACCAAGCGCAAGGGAATTAACGAATACATCAATCTGTATAGCCAGCAGATCAACGACAAAACTCTGAAGAAATACAAGATGTCTGTGCTGTTCAAACAGATCCTGAGTGATACCGAGTCCAAGTCTTTTGTCATTGATAAACTGGAAGATGACTCAGACGTGGTCACTACCATGCAGAGCTTTTATGAGCAGATCGCCGCTTTCAAGACAGTGGAGGAAAAATCTATTAAGGAAACTCTGAGTCTGCTGTTCGATGACCTGAAAGCCCAGAAGCTGGACCTGAGTAAGATCTACTTCAAAAACGATAAGAGTCTGACAGACCTGTCACAGCAGGTGTTTGATGACTATTCCGTGATTGGGACCGCCGTCCTGGAGTACATTACACAGCAGATCGCTCCAAAGAACCTGGATAATCCCTCTAAGAAAGAGCAGGAACTGATCGCTAAGAAAACCGAGAAGGCAAAATATCTGAGTCTGGAAACAATTAAGCTGGCACTGGAGGAGTTCAACAAGCACAGGGATATTGACAAACAGTGCCGCTTTGAGGAAATCCTGGCCAACTTCGCAGCCATCCCCATGATTTTTGATGAGATCGCCCAGAACAAAGACAATCTGGCTCAGATCAGTATTAAGTACCAGAACCAGGGCAAGAAAGACCTGCTGCAGGCTTCAGCAGAAGATGACGTGAAAGCCATCAAGGATCTGCTGGACCAGACCAACAATCTGCTGCACAAGCTGAAAATCTTCCATATTAGTCAGTCAGAGGATAAGGCTAATATCCTGGATAAAGACGAACACTTCTACCTGGTGTTCGAGGAATGTTACTTCGAGCTGGCAAACATTGTCCCCCTGTATAACAAGATTAGGAACTACATCACACAGAAGCCTTACTCTGACGAGAAGTTTAAACTGAACTTCGAAAATAGTACCCTGGCCAACGGGTGGGATAAGAACAAGGAGCCTGACAACACAGCTATCCTGTTCATCAAGGATGACAAGTACTATCTGGGAGTGATGAATAAGAAAAACAATAAGATCTTCGATGACAAAGCCATTAAGGAGAACAAAGGGGAAGGATACAAGAAAATCGTGTATAAGCTGCTGCCCGGCGCAAATAAGATGCTGCCTAAGGTGTTCTTCAGCGCCAAGAGTATCAAATTCTACAACCCATCCGAGGACATCCTGCGGATTAGAAATCACTCAACACATACTAAGAACGGGAGCCCCCAGAAGGGATATGAGAAATTTGAGTTCAACATCGAGGATTGCAGGAAGTTTATTGACTTCTACAAGCAGAGCATCTCCAAACACCCTGAATGGAAGGATTTTGGCTTCCGGTTTTCCGACACACAGAGATATAACTCTATCGACGAGTTCTACCGCGAGGTGGAAAATCAGGGGTATAAGCTGACTTTTGAGAACATTTCTGAAAGTTACATCGACAGCGTGGTCAATCAGGGAAAGCTGTACCTGTTCCAGATCTATAACAAAGATTTTTCAGCATACAGCAAGGGCAGACCAAACCTGCATACACTGTACTGGAAGGCCCTGTTCGATGAGAGGAATCTGCAGGACGTGGTCTATAAACTGAACGGAGAGGCCGAACTGTTTTACCGGAAGCAGTCTATTCCTAAGAAAATCACTCACCCAGCTAAGGAGGCCATCGCTAACAAGAACAAGGACAATCCTAAGAAAGAGAGCGTGTTCGAATACGATCTGATTAAGGACAAGCGGTTCACCGAAGATAAGTTCTTTTTCCATTGTCCAATCACCATTAACTTCAAGTCAAGCGGCGCTAACAAGTTCAACGACGAGATCAATCTGCTGCTGAAGGAAAAAGCAAACGATGTGCACATCCTGAGCATTGACCGAGGAGAGCGGCATCTGGCCTACTATACCCTGGTGGATGGCAAAGGGAATATCATTAAGCAGGATACATTCAACATCATTGGCAATGACCGGATGAAAACCAACTACCACGATAAACTGGCTGCAATCGAGAAGGATAGAGACTCAGCTAGGAAGGACTGGAAGAAAATCAACAACATTAAGGAGATGAAGGAAGGCTATCTGAGCCAGGTGGTCCATGAGATTGCAAAGCTGGTCATCGAATACAATGCCATTGTGGTGTTCGAGGATCTGAACTTCGGCTTTAAGAGGGGGCGCTTTAAGGTGGAAAAACAGGTCTATCAGAAGCTGGAGAAAATGCTGATCGAAAAGCTGAATTACCTGGTGTTTAAAGATAACGAGTTCGACAAGACCGGAGGCGTCCTGAGAGCCTACCAGCTGACAGCTCCCTTTGAAACTTTCAAGAAAATGGGAAAACAGACAGGCATCATCTACTATGTGCCAGCCGGATTCACTTCCAAGATCTGCCCCGTGACCGGCTTTGTCAACCAGCTGTACCCTAAATATGAGTCAGTGAGCAAGTCCCAGGAATTTTTCAGCAAGTTCGATAAGATCTGTTATAATCTGGACAAGGGGTACTTCGAGTTTTCCTTCGATTACAAGAACTTCGGCGACAAGGCCGCTAAGGGGAAATGGACCATTGCCTCCTTCGGATCTCGCCTGATCAACTTTCGAAATTCCGATAAAAACCACAATTGGGACACTAGGGAGGTGTACCCAACCAAGGAGCTGGAAAAGCTGCTGAAAGACTACTCTATCGAGTATGGACATGGCGAATGCATCAAGGCAGCCATCTGTGGCGAGAGTGATAAGAAATTTTTCGCCAAGCTGACCTCAGTGCTGAATACAATCCTGCAGATGCGGAACTCAAAGACCGGGACAGAACTGGACTATCTGATTAGCCCCGTGGCTGATGTCAACGGAAACTTCTTCGACAGCAGACAGGCACCCAAAAATATGCCTCAGGATGCAGACGCCAACGGGGCCTACCACATCGGGCTGAAGGGACTGATGCTGCTGGGCCGGATCAAGAACAATCAGGAGGGGAAGAAGCTGAACCTGGTCATTAAGAACGAGGAATACTTCGAGTTTGTCCAGAATAGAAATAACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC5- Peregrinibacteria bacterium GW2011_GWA_33_10 (PeCpfl)(SEQ ID NO: 217) ATGTCCAACTTCTTTAAGAATTTCACCAACCTGTATGAGCTGTCCAAGACACTGAGGTTTGAGCTGAAGCCCGTGGGCGACACCCTGACAAACATGAAGGACCACCTGGAGTACGATGAGAAGCTGCAGACCTTCCTGAAGGATCAGAATATCGACGATGCCTATCAGGCCCTGAAGCCTCAGTTCGACGAGATCCACGAGGAGTTTATCACAGATTCTCTGGAGAGCAAGAAGGCCAAGGAGATCGACTTCTCCGAGTACCTGGATCTGTTTCAGGAGAAGAAGGAGCTGAACGACTCTGAGAAGAAGCTGCGCAACAAGATCGGCGAGACATTCAACAAGGCCGGCGAGAAGTGGAAGAAGGAGAAGTACCCTCAGTATGAGTGGAAGAAGGGCTCCAAGATCGCCAATGGCGCCGACATCCTGTCTTGCCAGGATATGCTGCAGTTTATCAAGTATAAGAACCCAGAGGATGAGAAGATCAAGAATTACATCGACGATACACTGAAGGGCTTCTTTACCTATTTCGGCGGCTTTAATCAGAACAGGGCCAACTACTATGAGACAAAGAAGGAGGCCTCCACCGCAGTGGCAACAAGGATCGTGCACGAGAACCTGCCAAAGTTCTGTGACAATGTGATCCAGTTTAAGCACATCATCAAGCGGAAGAAGGATGGCACCGTGGAGAAAACCGAGAGAAAGACCGAGTACCTGAACGCCTACCAGTATCTGAAGAACAATAACAAGATCACACAGATCAAGGACGCCGAGACAGAGAAGATGATCGAGTCTACACCCATCGCCGAGAAGATCTTCGACGTGTACTACTTCAGCAGCTGCCTGAGCCAGAAGCAGATCGAGGAGTACAACCGGATCATCGGCCACTATAATCTGCTGATCAACCTGTATAACCAGGCCAAGAGATCTGAGGGCAAGCACCTGAGCGCCAACGAGAAGAAGTATAAGGACCTGCCTAAGTTCAAGACCCTGTATAAGCAGATCGGCTGCGGCAAGAAGAAGGACCTGTTTTACACAATCAAGTGTGATACCGAGGAGGAGGCCAATAAGTCCCGGAACGAGGGCAAGGAGTCCCACTCTGTGGAGGAGATCATCAACAAGGCCCAGGAGGCCATCAATAAGTACTTCAAGTCTAATAACGACTGTGAGAATATCAACACCGTGCCCGACTTCATCAACTATATCCTGACAAAGGAGAATTACGAGGGCGTGTATTGGAGCAAGGCCGCCATGAACACCATCTCCGACAAGTACTTCGCCAATTATCACGACCTGCAGGATAGACTGAAGGAGGCCAAGGTGTTTCAGAAGGCCGATAAGAAGTCCGAGGACGATATCAAGATCCCAGAGGCCATCGAGCTGTCTGGCCTGTTCGGCGTGCTGGACAGCCTGGCCGATTGGCAGACCACACTGTTTAAGTCTAGCATCCTGAGCAACGAGGACAAGCTGAAGATCATCACAGATTCCCAGACCCCCTCTGAGGCCCTGCTGAAGATGATCTTCAATGACATCGAGAAGAACATGGAGTCCTTTCTGAAGGAGACAAACGATATCATCACCCTGAAGAAGTATAAGGGCAATAAGGAGGGCACCGAGAAGATCAAGCAGTGGTTCGACTATACACTGGCCATCAACCGGATGCTGAAGTACTTTCTGGTGAAGGAGAATAAGATCAAGGGCAACTCCCTGGATACCAATATCTCTGAGGCCCTGAAAACCCTGATCTACAGCGACGATGCCGAGTGGTTCAAGTGGTACGACGCCCTGAGAAACTATCTGACCCAGAAGCCTCAGGATGAGGCCAAGGAGAATAAGCTGAAGCTGAATTTCGACAACCCATCTCTGGCCGGCGGCTGGGATGTGAACAAGGAGTGCAGCAATTTTTGCGTGATCCTGAAGGACAAGAACGAGAAGAAGTACCTGGCCATCATGAAGAAGGGCGAGAATACCCTGTTCCAGAAGGAGTGGACAGAGGGCCGGGGCAAGAACCTGACAAAGAAGTCTAATCCACTGTTCGAGATCAATAACTGCGAGATCCTGAGCAAGATGGAGTATGACTTTTGGGCCGACGTGAGCAAGATGATCCCCAAGTGTAGCACCCAGCTGAAGGCCGTGGTGAACCACTTCAAGCAGTCCGACAATGAGTTCATCTTTCCTATCGGCTACAAGGTGACAAGCGGCGAGAAGTTTAGGGAGGAGTGCAAGATCTCCAAGCAGGACTTCGAGCTGAATAACAAGGTGTTTAATAAGAACGAGCTGAGCGTGACCGCCATGCGCTACGATCTGTCCTCTACACAGGAGAAGCAGTATATCAAGGCCTTCCAGAAGGAGTACTGGGAGCTGCTGTTTAAGCAGGAGAAGCGGGACACCAAGCTGACAAATAACGAGATCTTCAACGAGTGGATCAATTTTTGCAACAAGAAGTATAGCGAGCTGCTGTCCTGGGAGAGAAAGTACAAGGATGCCCTGACCAATTGGATCAACTTCTGTAAGTACTTTCTGAGCAAGTATCCCAAGACCACACTGTTCAACTACTCTTTTAAGGAGAGCGAGAATTATAACTCCCTGGACGAGTTCTACCGGGACGTGGATATCTGTTCTTACAAGCTGAATATCAACACCACAATCAATAAGAGCATCCTGGATAGACTGGTGGAGGAGGGCAAGCTGTACCTGTTTGAGATCAAGAATCAGGACAGCAACGATGGCAAGTCCATCGGCCACAAGAATAACCTGCACACCATCTACTGGAACGCCATCTTCGAGAATTTTGACAACAGGCCTAAGCTGAATGGCGAGGCCGAGATCTTCTATCGCAAGGCCATCTCCAAGGATAAGCTGGGCATCGTGAAGGGCAAGAAAACCAAGAACGGCACCGAGATCATCAAGAATTACAGATTCAGCAAGGAGAAGTTTATCCTGCACGTGCCAATCACCCTGAACTTCTGCTCCAATAACGAGTATGTGAATGACATCGTGAACACAAAGTTCTACAATTTTTCCAACCTGCACTTTCTGGGCATCGATAGGGGCGAGAAGCACCTGGCCTACTATTCTCTGGTGAATAAGAACGGCGAGATCGTGGACCAGGGCACACTGAACCTGCCTTTCACCGACAAGGATGGCAATCAGCGCAGCATCAAGAAGGAGAAGTACTTTTATAACAAGCAGGAGGACAAGTGGGAGGCCAAGGAGGTGGATTGTTGGAATTATAACGACCTGCTGGATGCCATGGCCTCTAACCGGGACATGGCCAGAAAGAATTGGCAGAGGATCGGCACCATCAAGGAGGCCAAGAACGGCTACGTGAGCCTGGTCATCAGGAAGATCGCCGATCTGGCCGTGAATAACGAGCGCCCCGCCTTCATCGTGCTGGAGGACCTGAATACAGGCTTTAAGCGGTCCAGACAGAAGATCGATAAGAGCGTGTACCAGAAGTTCGAGCTGGCCCTGGCCAAGAAGCTGAACTTTCTGGTGGACAAGAATGCCAAGCGCGATGAGATCGGCTCCCCTACAAAGGCCCTGCAGCTGACCCCCCCTGTGAATAACTACGGCGACATTGAGAACAAGAAGCAGGCCGGCATCATGCTGTATACCCGGGCCAATTATACCTCTCAGACAGATCCAGCCACAGGCTGGAGAAAGACCATCTATCTGAAGGCCGGCCCCGAGGAGACAACATACAAGAAGGACGGCAAGATCAAGAACAAGAGCGTGAAGGACCAGATCATCGAGACATTCACCGATATCGGCTTTGACGGCAAGGATTACTATTTCGAGTACGACAAGGGCGAGTTTGTGGATGAGAAAACCGGCGAGATCAAGCCCAAGAAGTGGCGGCTGTACTCCGGCGAGAATGGCAAGTCCCTGGACAGGTTCCGCGGAGAGAGGGAGAAGGATAAGTATGAGTGGAAGATCGACAAGATCGATATCGTGAAGATCCTGGACGATCTGTTCGTGAATTTTGACAAGAACATCAGCCTGCTGAAGCAGCTGAAGGAGGGCGTGGAGCTGACCCGGAATAACGAGCACGGCACAGGCGAGTCCCTGAGATTCGCCATCAACCTGATCCAGCAGATCCGGAATACCGGCAATAACGAGAGAGACAACGATTTCATCCTGTCCCCAGTGAGGGACGAGAATGGCAAGCACTTTGACTCTCGCGAGTACTGGGATAAGGAGACAAAGGGCGAGAAGATCAGCATGCCCAGCTCCGGCGATGCCAATGGCGCCTTCAACATCGCCCGGAAGGGCATCATCATGAACGCCCACATCCTGGCCAATAGCGACTCCAAGGATCTGTCCCTGTTCGTGTCTGACGAGGAGTGGGATCTGCACCTGAATAACAAGACCGAGTGGAAGAAGCAGCTGAACATCTTTTCTAGCAGGAAGGCCATGGCCAAGCGCAAGAAGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTC6- Parcubacteria bacterium GWC2011_GWC2_44_17 (PbCpfl) (SEQ ID NO: 218)ATGGAGAACATCTTCGACCAGTTTATCGGCAAGTACAGCCTGTCCAAGACCCTGAGATTCGAGCTGAAGCCCGTGGGCAAGACAGAGGACTTCCTGAAGATCAACAAGGTGTTTGAGAAGGATCAGACCATCGACGATAGCTACAATCAGGCCAAGTTCTATTTTGATTCCCTGCACCAGAAGTTTATCGACGCCGCCCTGGCCTCCGATAAGACATCCGAGCTGTCTTTCCAGAACTTTGCCGACGTGCTGGAGAAGCAGAATAAGATCATCCTGGATAAGAAGAGAGAGATGGGCGCCCTGAGGAAGCGCGACAAGAACGCCGTGGGCATCGATAGGCTGCAGAAGGAGATCAATGACGCCGAGGATATCATCCAGAAGGAGAAGGAGAAGATCTACAAGGACGTGCGCACCCTGTTCGATAACGAGGCCGAGTCTTGGAAAACCTACTATCAGGAGCGGGAGGTGGACGGCAAGAAGATCACCTTCAGCAAGGCCGACCTGAAGCAGAAGGGCGCCGATTTTCTGACAGCCGCCGGCATCCTGAAGGTGCTGAAGTATGAGTTCCCCGAGGAGAAGGAGAAGGAGTTTCAGGCCAAGAACCAGCCCTCCCTGTTCGTGGAGGAGAAGGAGAATCCTGGCCAGAAGAGGTACATCTTCGACTCTTTTGATAAGTTCGCCGGCTATCTGACCAAGTTTCAGCAGACAAAGAAGAATCTGTACGCAGCAGACGGCACCAGCACAGCAGTGGCCACCCGCATCGCCGATAACTTTATCATCTTCCACCAGAATACCAAGGTGTTCCGGGACAAGTACAAGAACAATCACACAGACCTGGGCTTCGATGAGGAGAACATCTTTGAGATCGAGAGGTATAAGAATTGCCTGCTGCAGCGCGAGATCGAGCACATCAAGAATGAGAATAGCTACAACAAGATCATCGGCCGGATCAATAAGAAGATCAAGGAGTATCGGGACCAGAAGGCCAAGGATACCAAGCTGACAAAGTCCGACTTCCCTTTCTTTAAGAACCTGGATAAGCAGATCCTGGGCGAGGTGGAGAAGGAGAAGCAGCTGATCGAGAAAACCCGGGAGAAAACCGAGGAGGACGTGCTGATCGAGCGGTTCAAGGAGTTCATCGAGAACAATGAGGAGAGGTTCACCGCCGCCAAGAAGCTGATGAATGCCTTCTGTAACGGCGAGTTTGAGTCCGAGTACGAGGGCATCTATCTGAAGAATAAGGCCATCAACACAATCTCCCGGAGATGGTTCGTGTCTGACAGAGATTTTGAGCTGAAGCTGCCTCAGCAGAAGTCCAAGAACAAGTCTGAGAAGAATGAGCCAAAGGTGAAGAAGTTCATCTCCATCGCCGAGATCAAGAACGCCGTGGAGGAGCTGGACGGCGATATCTTTAAGGCCGTGTTCTACGACAAGAAGATCATCGCCCAGGGCGGCTCTAAGCTGGAGCAGTTCCTGGTCATCTGGAAGTACGAGTTTGAGTATCTGTTCCGGGACATCGAGAGAGAGAACGGCGAGAAGCTGCTGGGCTATGATAGCTGCCTGAAGATCGCCAAGCAGCTGGGCATCTTCCCACAGGAGAAGGAGGCCCGCGAGAAGGCAACCGCCGTGATCAAGAATTACGCCGACGCCGGCCTGGGCATCTTCCAGATGATGAAGTATTTTTCTCTGGACGATAAGGATCGGAAGAACACCCCCGGCCAGCTGAGCACAAATTTCTACGCCGAGTATGACGGCTACTACAAGGATTTCGAGTTTATCAAGTACTACAACGAGTTTAGGAACTTCATCACCAAGAAGCCTTTCGACGAGGATAAGATCAAGCTGAACTTTGAGAATGGCGCCCTGCTGAAGGGCTGGGACGAGAACAAGGAGTACGATTTCATGGGCGTGATCCTGAAGAAGGAGGGCCGCCTGTATCTGGGCATCATGCACAAGAACCACCGGAAGCTGTTTCAGTCCATGGGCAATGCCAAGGGCGACAACGCCAATAGATACCAGAAGATGATCTATAAGCAGATCGCCGACGCCTCTAAGGATGTGCCCAGGCTGCTGCTGACCAGCAAGAAGGCCATGGAGAAGTTCAAGCCTTCCCAGGAGATCCTGAGAATCAAGAAGGAGAAAACCTTCAAGCGGGAGAGCAAGAACTTTTCCCTGAGAGATCTGCACGCCCTGATCGAGTACTATAGGAACTGCATCCCTCAGTACAGCAATTGGTCCTTTTATGACTTCCAGTTTCAGGATACCGGCAAGTACCAGAATATCAAGGAGTTCACAGACGATGTGCAGAAGTACGGCTATAAGATCTCCTTTCGCGACATCGACGATGAGTATATCAATCAGGCCCTGAACGAGGGCAAGATGTACCTGTTCGAGGTGGTGAACAAGGATATCTATAACACCAAGAATGGCTCCAAGAATCTGCACACACTGTACTTTGAGCACATCCTGTCTGCCGAGAACCTGAATGACCCAGTGTTCAAGCTGTCTGGCATGGCCGAGATCTTTCAGCGGCAGCCCAGCGTGAACGAAAGAGAGAAGATCACCACACAGAAGAATCAGTGTATCCTGGACAAGGGCGATAGAGCCTACAAGTATAGGCGCTACACCGAGAAGAAGATCATGTTCCACATGAGCCTGGTGCTGAACACAGGCAAGGGCGAGATCAAGCAGGTGCAGTTTAATAAGATCATCAACCAGAGGATCAGCTCCTCTGACAACGAGATGAGGGTGAATGTGATCGGCATCGATCGCGGCGAGAAGAACCTGCTGTACTATAGCGTGGTGAAGCAGAATGGCGAGATCATCGAGCAGGCCTCCCTGAACGAGATCAATGGCGTGAACTACCGGGACAAGCTGATCGAGAGGGAGAAGGAGCGCCTGAAGAACCGGCAGAGCTGGAAGCCTGTGGTGAAGATCAAGGATCTGAAGAAGGGCTACATCTCCCACGTGATCCACAAGATCTGCCAGCTGATCGAGAAGTATTCTGCCATCGTGGTGCTGGAGGACCTGAATATGAGATTCAAGCAGATCAGGGGAGGAATCGAGCGGAGCGTGTACCAGCAGTTCGAGAAGGCCCTGATCGATAAGCTGGGCTATCTGGTGTTTAAGGACAACAGGGATCTGAGGGCACCAGGAGGCGTGCTGAATGGCTACCAGCTGTCTGCCCCCTTTGTGAGCTTCGAGAAGATGCGCAAGCAGACCGGCATCCTGTTCTACACACAGGCCGAGTATACCAGCAAGACAGACCCAATCACCGGCTTTCGGAAGAACGTGTATATCTCTAATAGCGCCTCCCTGGATAAGATCAAGGAGGCCGTGAAGAAGTTCGACGCCATCGGCTGGGATGGCAAGGAGCAGTCTTACTTCTTTAAGTACAACCCTTACAACCTGGCCGACGAGAAGTATAAGAACTCTACCGTGAGCAAGGAGTGGGCCATCTTTGCCAGCGCCCCAAGAATCCGGAGACAGAAGGGCGAGGACGGCTACTGGAAGTATGATAGGGTGAAAGTGAATGAGGAGTTCGAGAAGCTGCTGAAGGTCTGGAATTTTGTGAACCCAAAGGCCACAGATATCAAGCAGGAGATCATCAAGAAGGAGAAGGCAGGCGACCTGCAGGGAGAGAAGGAGCTGGATGGCCGGCTGAGAAACTTTTGGCACTCTTTCATCTACCTGTTTAACCTGGTGCTGGAGCTGCGCAATTCTTTCAGCCTGCAGATCAAGATCAAGGCAGGAGAAGTGATCGCAGTGGACGAGGGCGTGGACTTCATCGCCAGCCCAGTGAAGCCCTTCTTTACCACACCCAACCCTTACATCCCCTCCAACCTGTGCTGGCTGGCCGTGGAGAATGCAGACGCAAACGGAGCCTATAATATCGCCAGGAAGGGCGTGATGATCCTGAAGAAGATCCGCGAGCACGCCAAGAAGGACCCCGAGTTCAAGAAGCTGCCAAACCTGTTTATCAGCAATGCAGAGTGGGACGAGGCAGCCCGGGATTGGGGCAAGTACGCAGGCACCACAGCCCTGAACCTGGACCACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCCTACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTC7- Smithella sp. SC_K08D17 (SsCpfl) (SEQ ID NO: 219)ATGCAGACCCTGTTTGAGAACTTCACAAATCAGTACCCAGTGTCCAAGACCCTGCGCTTTGAGCTGATCCCCCAGGGCAAGACAAAGGACTTCATCGAGCAGAAGGGCCTGCTGAAGAAGGATGAGGACCGGGCCGAGAAGTATAAGAAGGTGAAGAACATCATCGATGAGTACCACAAGGACTTCATCGAGAAGTCTCTGAATGGCCTGAAGCTGGACGGCCTGGAGAAGTACAAGACCCTGTATCTGAAGCAGGAGAAGGACGATAAGGATAAGAAGGCCTTTGACAAGGAGAAGGAGAACCTGCGCAAGCAGATCGCCAATGCCTTCCGGAACAATGAGAAGTTTAAGACACTGTTCGCCAAGGAGCTGATCAAGAACGATCTGATGTCTTTCGCCTGCGAGGAGGACAAGAAGAATGTGAAGGAGTTTGAGGCCTTCACCACATACTTCACCGGCTTCCACCAGAACCGCGCCAATATGTACGTGGCCGATGAGAAGAGAACAGCCATCGCCAGCAGGCTGATCCACGAGAACCTGCCAAAGTTTATCGACAATATCAAGATCTTCGAGAAGATGAAGAAGGAGGCCCCCGAGCTGCTGTCTCCTTTCAACCAGACCCTGAAGGATATGAAGGACGTGATCAAGGGCACCACACTGGAGGAGATCTTTAGCCTGGATTATTTCAACAAGACCCTGACACAGAGCGGCATCGACATCTACAATTCCGTGATCGGCGGCAGAACCCCTGAGGAGGGCAAGACAAAGATCAAGGGCCTGAACGAGTACATCAATACCGACTTCAACCAGAAGCAGACAGACAAGAAGAAGCGGCAGCCAAAGTTCAAGCAGCTGTATAAGCAGATCCTGAGCGATAGGCAGAGCCTGTCCTTTATCGCCGAGGCCTTCAAGAACGACACCGAGATCCTGGAGGCCATCGAGAAGTTTTACGTGAATGAGCTGCTGCACTTCAGCAATGAGGGCAAGTCCACAAACGTGCTGGACGCCATCAAGAATGCCGTGTCTAACCTGGAGAGCTTTAACCTGACCAAGATGTATTTCCGCTCCGGCGCCTCTCTGACAGACGTGAGCCGGAAGGTGTTTGGCGAGTGGAGCATCATCAATAGAGCCCTGGACAACTACTATGCCACCACATATCCAATCAAGCCCAGAGAGAAGTCTGAGAAGTACGAGGAGAGGAAGGAGAAGTGGCTGAAGCAGGACTTCAACGTGAGCCTGATCCAGACCGCCATCGATGAGTACGACAACGAGACAGTGAAGGGCAAGAACAGCGGCAAAGTGATCGCCGATTATTTTGCCAAGTTCTGCGACGATAAGGAGACAGACCTGATCCAGAAGGTGAACGAGGGCTACATCGCCGTGAAGGATCTGCTGAATACACCCTGTCCTGAGAACGAGAAGCTGGGCAGCAATAAGGACCAGGTGAAGCAGATCAAGGCCTTTATGGATTCTATCATGGACATCATGCACTTCGTGCGCCCCCTGAGCCTGAAGGATACCGACAAGGAGAAGGATGAGACATTCTACTCCCTGTTCACACCTCTGTACGACCACCTGACCCAGACAATCGCCCTGTATAACAAGGTGCGGAACTATCTGACCCAGAAGCCTTACAGCACAGAGAAGATCAAGCTGAACTTCGAGAACAGCACCCTGCTGGGCGGCTGGGATCTGAATAAGGAGACAGACAACACAGCCATCATCCTGAGGAAGGATAACCTGTACTATCTGGGCATCATGGACAAGAGGCACAATCGCATCTTTCGGAACGTGCCCAAGGCCGATAAGAAGGACTTCTGCTACGAGAAGATGGTGTATAAGCTGCTGCCTGGCGCCAACAAGATGCTGCCAAAGGTGTTCTTTTCTCAGAGCAGAATCCAGGAGTTTACCCCTTCCGCCAAGCTGCTGGAGAACTACGCCAATGAGACACACAAGAAGGGCGATAATTTCAACCTGAATCACTGTCACAAGCTGATCGATTTCTTTAAGGACTCTATCAACAAGCACGAGGATTGGAAGAATTTCGACTTTAGGTTCAGCGCCACCTCCACCTACGCCGACCTGAGCGGCTTTTACCACGAGGTGGAGCACCAGGGCTACAAGATCTCTTTTCAGAGCGTGGCCGATTCCTTCATCGACGATCTGGTGAACGAGGGCAAGCTGTACCTGTTCCAGATCTATAATAAGGACTTTTCCCCATTCTCTAAGGGCAAGCCCAACCTGCACACCCTGTACTGGAAGATGCTGTTTGATGAGAACAATCTGAAGGACGTGGTGTATAAGCTGAATGGCGAGGCCGAGGTGTTCTACCGCAAGAAGAGCATTGCCGAGAAGAACACCACAATCCACAAGGCCAATGAGTCCATCATCAACAAGAATCCTGATAACCCAAAGGCCACCAGCACCTTCAACTATGATATCGTGAAGGACAAGAGATACACCATCGACAAGTTTCAGTTCCACATCCCAATCACAATGAACTTTAAGGCCGAGGGCATCTTCAACATGAATCAGAGGGTGAATCAGTTCCTGAAGGCCAATCCCGATATCAACATCATCGGCATCGACAGAGGCGAGAGGCACCTGCTGTACTATGCCCTGATCAACCAGAAGGGCAAGATCCTGAAGCAGGATACCCTGAATGTGATCGCCAACGAGAAGCAGAAGGTGGACTACCACAATCTGCTGGATAAGAAGGAGGGCGACCGCGCAACCGCAAGGCAGGAGTGGGGCGTGATCGAGACAATCAAGGAGCTGAAGGAGGGCTATCTGTCCCAGGTCATCCACAAGCTGACCGATCTGATGATCGAGAACAATGCCATCATCGTGATGGAGGACCTGAACTTTGGCTTCAAGCGGGGCAGACAGAAGGTGGAGAAGCAGGTGTATCAGAAGTTTGAGAAGATGCTGATCGATAAGCTGAATTACCTGGTGGACAAGAATAAGAAGGCAAACGAGCTGGGAGGCCTGCTGAACGCATTCCAGCTGGCCAATAAGTTTGAGTCCTTCCAGAAGATGGGCAAGCAGAACGGCTTTATCTTCTACGTGCCCGCCTGGAATACCTCTAAGACAGATCCTGCCACCGGCTTTATCGACTTCCTGAAGCCCCGCTATGAGAACCTGAATCAGGCCAAGGATTTCTTTGAGAAGTTTGACTCTATCCGGCTGAACAGCAAGGCCGATTACTTTGAGTTCGCCTTTGACTTCAAGAATTTCACCGAGAAGGCCGATGGCGGCAGAACCAAGTGGACAGTGTGCACCACAAACGAGGACAGATATGCCTGGAATAGGGCCCTGAACAATAACAGGGGCAGCCAGGAGAAGTACGACATCACAGCCGAGCTGAAGTCCCTGTTCGATGGCAAGGTGGACTATAAGTCTGGCAAGGATCTGAAGCAGCAGATCGCCAGCCAGGAGTCCGCCGACTTCTTTAAGGCCCTGATGAAGAACCTGTCCATCACCCTGTCTCTGAGACACAATAACGGCGAGAAGGGCGATAATGAGCAGGACTACATCCTGTCCCCTGTGGCCGATTCTAAGGGCCGCTTCTTTGACTCCCGGAAGGCCGACGATGACATGCCAAAGAATGCCGACGCCAACGGCGCCTATCACATCGCCCTGAAGGGCCTGTGGTGTCTGGAGCAGATCAGCAAGACCGATGACCTGAAGAAGGTGAAGCTGGCCATCTCCAACAAGGAGTGGCTGGAGTTCGTGCAGACACTGAAGGGCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAA G GGATCCTACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTC8- Acidaminococcus sp. BV3L6 (AsCpfl) (SEQ ID NO: 20)ATGACACAGTTCGAGGGCTTTACCAACCTGTATCAGGTGAGCAAGACACTGCGGTTTGAGCTGATCCCACAGGGCAAGACCCTGAAGCACATCCAGGAGCAGGGCTTCATCGAGGAGGACAAGGCCCGCAATGATCACTACAAGGAGCTGAAGCCCATCATCGATCGGATCTACAAGACCTATGCCGACCAGTGCCTGCAGCTGGTGCAGCTGGATTGGGAGAACCTGAGCGCCGCCATCGACTCCTATAGAAAGGAGAAAACCGAGGAGACAAGGAACGCCCTGATCGAGGAGCAGGCCACATATCGCAATGCCATCCACGACTACTTCATCGGCCGGACAGACAACCTGACCGATGCCATCAATAAGAGACACGCCGAGATCTACAAGGGCCTGTTCAAGGCCGAGCTGTTTAATGGCAAGGTGCTGAAGCAGCTGGGCACCGTGACCACAACCGAGCACGAGAACGCCCTGCTGCGGAGCTTCGACAAGTTTACAACCTACTTCTCCGGCTTTTATGAGAACAGGAAGAACGTGTTCAGCGCCGAGGATATCAGCACAGCCATCCCACACCGCATCGTGCAGGACAACTTCCCCAAGTTTAAGGAGAATTGTCACATCTTCACACGCCTGATCACCGCCGTGCCCAGCCTGCGGGAGCACTTTGAGAACGTGAAGAAGGCCATCGGCATCTTCGTGAGCACCTCCATCGAGGAGGTGTTTTCCTTCCCTTTTTATAACCAGCTGCTGACACAGACCCAGATCGACCTGTATAACCAGCTGCTGGGAGGAATCTCTCGGGAGGCAGGCACCGAGAAGATCAAGGGCCTGAACGAGGTGCTGAATCTGGCCATCCAGAAGAATGATGAGACAGCCCACATCATCGCCTCCCTGCCACACAGATTCATCCCCCTGTTTAAGCAGATCCTGTCCGATAGGAACACCCTGTCTTTCATCCTGGAGGAGTTTAAGAGCGACGAGGAAGTGATCCAGTCCTTCTGCAAGTACAAGACACTGCTGAGAAACGAGAACGTGCTGGAGACAGCCGAGGCCCTGTT TAACGAGCTGAACAGCATCGACCTGACACACATCTTCATCAGCCACAAGAAGCTGGAGACAATCAGCAGCGCCCTGTGCGACCACTGGGATACACTGAGGAATGCCCTGTATGAGCGGAGAATCTCCGAGCTGACAGGCAAGATCACCAAGTCTGCCAAGGAGAAGGTGCAGCGCAGCCTGAAGCACGAGGATATCAACCTGCAGGAGATCATCTCTGCCGCAGGCAAGGAGCTGAGCGAGGCCTTCAAGCAGAAAACCAGCGAGATCCTGTCCCACGCACACGCCGCCCTGGATCAGCCACTGCCTACAACCCTGAAGAAGCAGGAGGAGAAGGAGATCCTGAAGTCTCAGCTGGACAGCCTGCTGGGCCTGTACCACCTGCTGGACTGGTTTGCCGTGGATGAGTCCAACGAGGTGGACCCCGAGTTCTCTGCCCGGCTGACCGGCATCAAGCTGGAGATGGAGCCTTCTCTGAGCTTCTACAACAAGGCCAGAAATTATGCCACCAAGAAGCCCTACTCCGTGGAGAAGTTCAAGCTGAACTTTCAGATGCCTACACTGGCCTCTGGCTGGGACGTGAATAAGGAGAAGAACAATGGCGCCATCCTGTTTGTGAAGAACGGCCTGTACTATCTGGGCATCATGCCAAAGCAGAAGGGCAGGTATAAGGCCCTGAGCTTCGAGCCCACAGAGAAAACCAGCGAGGGCTTTGATAAGATGTACTATGACTACTTCCCTGATGCCGCCAAGATGATCCCAAAGTGCAGCACCCAGCTGAAGGCCGTGACAGCCCACTTTCAGACCCACACAACCCCCATCCTGCTGTCCAACAATTTCATCGAGCCTCTGGAGATCACAAAGGAGATCTACGACCTGAACAATCCTGAGAAGGAGCCAAAGAAGTTTCAGACAGCCTACGCCAAGAAAACCGGCGACCAGAAGGGCTACAGAGAGGCCCTGTGCAAGTGGATCGACTTCACAAGGGATTTTCTGTCCAAGTATACCAAGACAACCTCTATCGATCTGTCTAGCCTGCGGCCATCCTCTCAGTATAAGGACCTGGGCGAGTACTATGCCGAGCTGAATCCCCTGCTGTACCACATCAGCTTCCAGAGAATCGCCGAGAAGGAGATCATGGATGCCGTGGAGACAGGCAAGCTGTACCTGTTCCAGATCTATAACAAGGACTTTGCCAAGGGCCACCACGGCAAGCCTAATCTGCACACACTGTATTGGACCGGCCTGTTTTCTCCAGAGAACCTGGCCAAGACAAGCATCAAGCTGAATGGCCAGGCCGAGCTGTTCTACCGCCCTAAGTCCAGGATGAAGAGGATGGCACACCGGCTGGGAGAGAAGATGCTGAACAAGAAGCTGAAGGATCAGAAAACCCCAATCCCCGACACCCTGTACCAGGAGCTGTACGACTATGTGAATCACAGACTGTCCCACGACCTGTCTGATGAGGCCAGGGCCCTGCTGCCCAACGTGATCACCAAGGAGGTGTCTCACGAGATCATCAAGGATAGGCGCTTTACCAGCGACAAGTTCTTTTTCCACGTGCCTATCACACTGAACTATCAGGCCGCCAATTCCCCATCTAAGTTCAACCAGAGGGTGAATGCCTACCTGAAGGAGCACCCCGAGACACCTATCATCGGCATCGATCGGGGCGAGAGAAACCTGATCTATATCACAGTGATCGACTCCACCGGCAAGATCCTGGAGCAGCGGAGCCTGAACACCATCCAGCAGTTTGATTACCAGAAGAAGCTGGACAACAGGGAGAAGGAGAGGGTGGCAGCAAGGCAGGCCTGGTCTGTGGTGGGCACAATCAAGGATCTGAAGCAGGGCTATCTGAGCCAGGTCATCCACGAGATCGTGGACCTGATGATCCACTACCAGGCCGTGGTGGTGCTGGAGAACCTGAATTTCGGCTTTAAGAGCAAGAGGACCGGCATCGCCGAGAAGGCCGTGTACCAGCAGTTCGAGAAGATGCTGATCGATAAGCTGAATTGCCTGGTGCTGAAGGACTATCCAGCAGAGAAAGTGGGAGGCGTGCTGAACCCATACCAGCTGACAGACCAGTTCACCTCCTTTGCCAAGATGGGCACCCAGTCTGGCTTCCTGTTTTACGTGCCTGCCCCATATACATCTAAGATCGATCCCCTGACCGGCTTCGTGGACCCCTTCGTGTGGAAAACCATCAAGAATCACGAGAGCCGCAAGCACTTCCTGGAGGGCTTCGACTTTCTGCACTACGACGTGAAAACCGGCGACTTCATCCTGCACTTTAAGATGAACAGAAATCTGTCCTTCCAGAGGGGCCTGCCCGGCTTTATGCCTGCATGGGATATCGTGTTCGAGAAGAACGAGACACAGTTTGACGCCAAGGGCACCCCTTTCATCGCCGGCAAGAGAATCGTGCCAGTGATCGAGAATCACAGATTCACCGGCAGATACCGGGACCTGTATCCTGCCAACGAGCTGATCGCCCTGCTGGAGGAGAAGGGCATCGTGTTCAGGGATGGCTCCAACATCCTGCCAAAGCTGCTGGAGAATGACGATTCTCACGCCATCGACACCATGGTGGCCCTGATCCGCAGCGTGCTGCAGATGCGGAACTCCAATGCCGCCACAGGCGAGGACTATATCAACAGCCCCGTGCGCGATCTGAATGGCGTGTGCTTCGACTCCCGGTTTCAGAACCCAGAGTGGCCCATGGACGCCGATGCCAATGGCGCCTACCACATCGCCCTGAAGGGCCAGCTGCTGCTGAATCACCTGAAGGAGAGCAAGGATCTGAAGCTGCAGAACGGCATCTCCAATCAGGACTGGCTGGCCTACATCCAGGAGCTGCGCAACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTC 9- Lachnospiraceae bacterium MA2020 (Lb2Cpf1)(SEQ ID NO: 221) ATGTACTATGAGTCCCTGACCAAGCAGTACCCCGTGTCTAAGACAATCCGGAATGAGCTGATCCCTATCGGCAAGACACTGGATAACATCCGCCAGAACAATATCCTGGAGAGCGACGTGAAGCGGAAGCAGAACTACGAGCACGTGAAGGGCATCCTGGATGAGTATCACAAGCAGCTGATCAACGAGGCCCTGGACAATTGCACCCTGCCATCCCTGAAGATCGCCGCCGAGATCTACCTGAAGAATCAGAAGGAGGTGTCTGACAGAGAGGATTTCAACAAGACACAGGACCTGCTGAGGAAGGAGGTGGTGGAGAAGCTGAAGGCCCACGAGAACTTTACCAAGATCGGCAAGAAGGACATCCTGGATCTGCTGGAGAAGCTGCCTTCCATCTCTGAGGACGATTACAATGCCCTGGAGAGCTTCCGCAACTTTTACACCTATTTCACATCCTACAACAAGGTGCGGGAGAATCTGTATTCTGATAAGGAGAAGAGCTCCACAGTGGCCTACAGACTGATCAACGAGAATTTCCCAAAGTTTCTGGACAATGTGAAGAGCTATAGGTTTGTGAAAACCGCAGGCATCCTGGCAGATGGCCTGGGAGAGGAGGAGCAGGACTCCCTGTTCATCGTGGAGACATTCAACAAGACCCTGACACAGGACGGCATCGATACCTACAATTCTCAAGTGGGCAAGATCAACTCTAGCATCAATCTGTATAACCAGAAGAATCAGAAGGCCAATGGCTTCAGAAAGATCCCCAAGATGAAGATGCTGTATAAGCAGATCCTGTCCGATAGGGAGGAGTCTTTCATCGACGAGTTTCAGAGCGATGAGGTGCTGATCGACAACGTGGAGTCTTATGGCAGCGTGCTGATCGAGTCTCTGAAGTCCTCTAAGGTGAGCGCCTTCTTTGATGCCCTGAGAGAGTCTAAGGGCAAGAACGTGTACGTGAAGAATGACCTGGCCAAGACAGCCATGAGCAACATCGTGTTCGAGAATTGGAGGACCTTTGACGATCTGCTGAACCAGGAGTACGACCTGGCCAACGAGAACAAGAAGAAGGACGATAAGTATTTCGAGAAGCGCCAGAAGGAGCTGAAGAAGAATAAGAGCTACTCCCTGGAGCACCTGTGCAACCTGTCCGAGGATTCTTGTAACCTGATCGAGAATTATATCCACCAGATCTCCGACGATATCGAGAATATCATCATCAACAATGAGACATTCCTGCGCATCGTGATCAATGAGCACGACAGGTCCCGCAAGCTGGCCAAGAACCGGAAGGCCGTGAAGGCCATCAAGGACTTTCTGGATTCTATCAAGGTGCTGGAGCGGGAGCTGAAGCTGATCAACAGCTCCGGCCAGGAGCTGGAGAAGGATCTGATCGTGTACTCTGCCCACGAGGAGCTGCTGGTGGAGCTGAAGCAGGTGGACAGCCTGTATAACATGACCAGAAATTATCTGACAAAGAAGCCTTTCTCTACCGAGAAGGTGAAGCTGAACTTTAATCGCAGCACACTGCTGAACGGCTGGGATCGGAATAAGGAGACAGACAACCTGGGCGTGCTGCTGCTGAAGGACGGCAAGTACTATCTGGGCATCATGAACACAAGCGCCAATAAGGCCTTCGTGAATCCCCCTGTGGCCAAGACCGAGAAGGTGTTTAAGAAGGTGGATTACAAGCTGCTGCCAGTGCCCAACCAGATGCTGCCAAAGGTGTTCTTTGCCAAGAGCAATATCGACTTCTATAACCCCTCTAGCGAGATCTACTCCAATTATAAGAAGGGCACCCACAAGAAGGGCAATATGTTTTCCCTGGAGGATTGTCACAACCTGATCGACTTCTTTAAGGAGTCTATCAGCAAGCACGAGGACTGGAGCAAGTTCGGCTTTAAGTTCAGCGATACAGCCTCCTACAACGACATCTCCGAGTTCTATCGCGAGGTGGAGAAGCAGGGCTACAAGCTGACCTATACAGACATCGATGAGACATACATCAATGATCTGATCGAGCGGAACGAGCTGTACCTGTTCCAGATCTATAATAAGGACTTTAGCATGTACTCCAAGGGCAAGCTGAACCTGCACACACTGTATTTCATGATGCTGTTTGATCAGCGCAATATCGACGACGTGGTGTATAAGCTGAACGGAGAGGCAGAGGTGTTCTATAGGCCAGCCTCCATCTCTGAGGACGAGCTGATCATCCACAAGGCCGGCGAGGAGATCAAGAACAAGAATCCTAACCGGGCCAGAACCAAGGAGACAAGCACCTTCAGCTACGACATCGTGAAGGATAAGCGGTATAGCAAGGATAAGTTTACCCTGCACATCCCCATCACAATGAACTTCGGCGTGGATGAGGTGAAGCGGTTCAACGACGCCGTGAACAGCGCCATCCGGATCGATGAGAATGTGAACGTGATCGGCATCGACCGGGGCGAGAGAAATCTGCTGTACGTGGTGGTCATCGACTCTAAGGGCAACATCCTGGAGCAGATCTCCCTGAACTCTATCATCAATAAGGAGTACGACATCGAGACAGATTATCACGCACTGCTGGATGAGAGGGAGGGCGGCAGAGATAAGGCCCGGAAGGACTGGAACACCGTGGAGAATATCAGGGACCTGAAGGCCGGCTACCTGAGCCAGGTGGTGAACGTGGTGGCCAAGCTGGTGCTGAAGTATAATGCCATCATCTGCCTGGAGGACCTGAACTTTGGCTTCAAGAGGGGCCGCCAGAAGGTGGAGAAGCAGGTGTACCAGAAGTTCGAGAAGATGCTGATCGATAAGCTGAATTACCTGGTCATCGACAAGAGCCGCGAGCAGACATCCCCTAAGGAGCTGGGAGGCGCCCTGAACGCACTGCAGCTGACCTCTAAGTTCAAGAGCTTTAAGGAGCTGGGCAAGCAGTCCGGCGTGATCTACTATGTGCCTGCCTACCTGACCTCTAAGATCGATCCAACCACAGGCTTCGCCAATCTGTTTTATATGAAGTGTGAGAACGTGGAGAAGTCCAAGAGATTCTTTGACGGCTTTGATTTCATCAGGTTCAACGCCCTGGAGAACGTGTTCGAGTTCGGCTTTGACTACCGGAGCTTCACCCAGAGGGCCTGCGGCATCAATTCCAAGTGGACCGTGTGCACCAACGGCGAGCGCATCATCAAGTATCGGAATCCAGATAAGAACAATATGTTCGACGAGAAGGTGGTGGTGGTGACCGATGAGATGAAGAACCTGTTTGAGCAGTACAAGATCCCCTATGAGGATGGCAGAAATGTGAAGGACATGATCATCAGCAACGAGGAGGCCGAGTTCTACCGGAGACTGTATAGGCTGCTGCAGCAGACCCTGCAGATGAGAAACAGCACCTCCGACGGCACAAGGGATTACATCATCTCCCCTGTGAAGAATAAGAGAGAGGCCTACTTCAACAGCGAGCTGTCCGACGGCTCTGTGCCAAAGGACGCCGATGCCAACGGCGCCTACAATATCGCCAGAAAGGGCCTGTGGGTGCTGGAGCAGATCAGGCAGAAGAGCGAGGGCGAGAAGATCAATCTGGCCATGACCAACGCCGAGTGGCTGGAGTATGCCCAGACACACCTGCTGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTC 10- Candidatus Methanoplasma termitum (CMtCpfl)(SEQ ID NO: 222) ATGAACAATTACGACGAGTTCACCAAGCTGTATCCTATCCAGAAAACCATCCGGTTTGAGCTGAAGCCACAGGGCAGAACCATGGAGCACCTGGAGACATTCAACTTCTTTGAGGAGGACCGGGATAGAGCCGAGAAGTATAAGATCCTGAAGGAGGCCATCGACGAGTACCACAAGAAGTTTATCGATGAGCACCTGACCAATATGTCCCTGGATTGGAACTCTCTGAAGCAGATCAGCGAGAAGTACTATAAGAGCAGGGAGGAGAAGGACAAGAAGGTGTTCCTGTCCGAGCAGAAGAGGATGCGCCAGGAGATCGTGTCTGAGTTTAAGAAGGACGATCGCTTCAAGGACCTGTTTTCCAAGAAGCTGTTCTCTGAGCTGCTGAAGGAGGAGATCTACAAGAAGGGCAACCACCAGGAGATCGACGCCCTGAAGAGCTTCGATAAGTTTTCCGGCTATTTCATCGGCCTGCACGAGAATAGGAAGAACATGTACTCCGACGGCGATGAGATCACCGCCATCTCCAATCGCATCGTGAATGAGAACTTCCCCAAGTTTCTGGATAACCTGCAGAAGTACCAGGAGGCCAGGAAGAAGTATCCTGAGTGGATCATCAAGGCCGAGAGCGCCCTGGTGGCCCACAATATCAAGATGGACGAGGTGTTCTCCCTGGAGTACTTTAATAAGGTGCTGAACCAGGAGGGCATCCAGCGGTACAACCTGGCCCTGGGCGGCTATGTGACCAAGAGCGGCGAGAAGATGATGGGCCTGAATGATGCCCTGAACCTGGCCCACCAGTCCGAGAAGAGCTCCAAGGGCAGAATCCACATGACCCCCCTGTTCAAGCAGATCCTGTCCGAGAAGGAGTCCTTCTCTTACATCCCCGACGTGTTTACAGAGGATTCTCAGCTGCTGCCTAGCATCGGCGGCTTCTTTGCCCAGATCGAGAATGACAAGGATGGCAACATCTTCGACCGGGCCCTGGAGCTGATCTCTAGCTACGCCGAGTATGATACCGAGCGGATCTATATCAGACAGGCCGACATCAATAGAGTGTCCAACGTGATCTTTGGAGAGTGGGGCACCCTGGGAGGCCTGATGAGGGAGTACAAGGCCGACTCTATCAATGATATCAACCTGGAGCGCACATGCAAGAAGGTGGACAAGTGGCTGGATTCTAAGGAGTTTGCCCTGAGCGATGTGCTGGAGGCCATCAAGAGGACCGGCAACAATGACGCCTTCAACGAGTATATCTCCAAGATGCGGACAGCCAGAGAGAAGATCGATGCCGCCCGCAAGGAGATGAAGTTCATCAGCGAGAAGATCTCCGGCGATGAGGAGTCTATCCACATCATCAAGACCCTGCTGGACAGCGTGCAGCAGTTCCTGCACTTCTTTAATCTGTTTAAGGCAAGGCAGGACATCCCACTGGATGGAGCCTTCTACGCCGAGTTTGACGAGGTGCACAGCAAGCTGTTTGCCATCGTGCCCCTGTATAACAAGGTGCGGAACTATCTGACCAAGAACAATCTGAACACAAAGAAGATCAAGCTGAATTTCAAGAACCCTACACTGGCCAATGGCTGGGACCAGAACAAGGTGTACGATTATGCCTCCCTGATCTTTCTGCGGGACGGCAATTACTATCTGGGCATCATCAATCCTAAGAGAAAGAAGAACATCAAGTTCGAGCAGGGCTCTGGCAACGGCCCCTTCTACCGGAAGATGGTGTATAAGCAGATCCCCGGCCCTAATAAGAACCTGCCAAGAGTGTTCCTGACCTCCACAAAGGGCAAGAAGGAGTATAAGCCCTCTAAGGAGATCATCGAGGGCTACGAGGCCGACAAGCACATCAGGGGCGATAAGTTCGACCTGGATTTTTGTCACAAGCTGATCGATTTCTTTAAGGAGTCCATCGAGAAGCACAAGGACTGGTCTAAGTTCAACTTCTACTTCAGCCCAACCGAGAGCTATGGCGACATCTCTGAGTTCTACCTGGATGTGGAGAAGCAGGGCTATCGCATGCACTTTGAGAATATCAGCGCCGAGACAATCGACGAGTATGTGGAGAAGGGCGATCTGTTTCTGTTCCAGATCTACAACAAGGATTTTGTGAAGGCCGCCACCGGCAAGAAGGACATGCACACAATCTACTGGAATGCCGCCTTCAGCCCCGAGAACCTGCAGGACGTGGTGGTGAAGCTGAACGGCGAGGCCGAGCTGTTTTATAGGGACAAGTCCGATATCAAGGAGATCGTGCACCGCGAGGGCGAGATCCTGGTGAATAGGACCTACAACGGCCGCACACCAGTGCCCGACAAGATCCACAAGAAGCTGACCGATTATCACAATGGCCGGACAAAGGACCTGGGCGAGGCCAAGGAGTACCTGGATAAGGTGAGATACTTCAAGGCCCACTATGACATCACCAAGGATCGGAGATACCTGAACGACAAGATCTATTTCCACGTGCCTCTGACCCTGAACTTCAAGGCCAACGGCAAGAAGAATCTGAACAAGATGGTCATCGAGAAGTTCCTGTCCGATGAGAAGGCCCACATCATCGGCATCGACAGGGGCGAGCGCAATCTGCTGTACTATTCCATCATCGACAGGTCTGGCAAGATCATCGATCAGCAGAGCCTGAATGTGATCGACGGCTTTGATTATCGGGAGAAGCTGAACCAGAGAGAGATCGAGATGAAGGATGCCCGCCAGTCTTGGAACGCCATCGGCAAGATCAAGGACCTGAAGGAGGGCTACCTGAGCAAGGCCGTGCACGAGATCACCAAGATGGCCATCCAGTATAATGCCATCGTGGTCATGGAGGAGCTGAACTACGGCTTCAAGCGGGGCCGGTTCAAGGTGGAGAAGCAGATCTATCAGAAGTTCGAGAATATGCTGATCGATAAGATGAACTACCTGGTGTTTAAGGACGCACCTGATGAGTCCCCAGGAGGCGTGCTGAATGCCTACCAGCTGACAAACCCACTGGAGTCTTTCGCCAAGCTGGGCAAGCAGACCGGCATCCTGTTTTACGTGCCAGCCGCCTATACATCCAAGATCGACCCCACCACAGGCTTCGTGAATCTGTTTAACACCTCCTCTAAGACAAACGCCCAGGAGCGGAAGGAGTTCCTGCAGAAGTTTGAGAGCATCTCCTATTCTGCCAAGGATGGCGGCATCTTTGCCTTCGCCTTTGACTACAGAAAGTTCGGCACCAGCAAGACAGATCACAAGAACGTGTGGACCGCCTATACAAACGGCGAGAGGATGCGCTACATCAAGGAGAAGAAGCGGAATGAGCTGTTTGACCCTTCTAAGGAGATCAAGGAGGCCCTGACCAGCTCCGGCATCAAGTACGATGGCGGCCAGAACATCCTGCCAGACATCCTGAGGAGCAACAATAACGGCCTGATCTACACAATGTATTCTAGCTTCATCGCCGCCATCCAGATGCGCGTGTACGACGGCAAGGAGGATTATATCATCAGCCCCATCAAGAACTCCAAGGGCGAGTTCTTTAGGACCGACCCCAAGAGGCGCGAGCTGCCTATCGACGCCGATGCCAATGGCGCCTACAACATCGCCCTGAGGGGAGAGCTGACAATGAGGGCAATCGCAGAGAAGTTCGACCCTGATAGCGAGAAGATGGCCAAGCTGGAGCTGAAGCACAAGGATTGGTTCGAGTTTATGCAGACCAGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATT C11- Eubacterium eligens (EeCpfl) (SEQ ID NO: 223)GAACGGCAATAGGTCCATCGTGTACCGCGAGTTCGTGGGCGTGATCCCCGTGGCCAAGACCCTGAGGAATGAGCTGCGCCCTGTGGGCCACACACAGGAGCACATCATCCAGAACGGCCTGATCCAGGAGGACGAGCTGCGGCAGGAGAAGAGCACCGAGCTGAAGAACATCATGGACGATTACTATAGAGAGTACATCGATAAGTCTCTGAGCGGCGTGACCGACCTGGACTTCACCCTGCTGTTCGAGCTGATGAACCTGGTGCAGAGCTCCCCCTCCAAGGACAATAAGAAGGCCCTGGAGAAGGAGCAGTCTAAGATGAGGGAGCAGATCTGCACCCACCTGCAGTCCGACTCTAACTACAAGAATATCTTTAACGCCAAGCTGCTGAAGGAGATCCTGCCTGATTTCATCAAGAACTACAATCAGTATGACGTGAAGGATAAGGCCGGCAAGCTGGAGACACTGGCCCTGTTTAATGGCTTCAGCACATACTTTACCGACTTCTTTGAGAAGAGGAAGAACGTGTTCACCAAGGAGGCCGTGAGCACATCCATCGCCTACCGCATCGTGCACGAGAACTCCCTGATCTTCCTGGCCAATATGACCTCTTATAAGAAGATCAGCGAGAAGGCCCTGGATGAGATCGAAGTGATCGAGAAGAACAATCAGGACAAGATGGGCGATTGGGAGCTGAATCAGATCTTTAACCCTGACTTCTACAATATGGTGCTGATCCAGTCCGGCATCGACTTCTACAACGAGATCTGCGGCGTGGTGAATGCCCACATGAACCTGTACTGTCAGCAGACCAAGAACAATTATAACCTGTTCAAGATGCGGAAGCTGCACAAGCAGATCCTGGCCTACACCAGCACCAGCTTCGAGGTGCCCAAGATGTTCGAGGACGATATGAGCGTGTATAACGCCGTGAACGCCTTCATCGACGAGACAGAGAAGGGCAACATCATCGGCAAGCTGAAGGATATCGTGAATAAGTACGACGAGCTGGATGAGAAGAGAATCTATATCAGCAAGGACTTTTACGAGACACTGAGCTGCTTCATGTCCGGCAACTGGAATCTGATCACAGGCTGCGTGGAGAACTTCTACGATGAGAACATCCACGCCAAGGGCAAGTCCAAGGAGGAGAAGGTGAAGAAGGCCGTGAAGGAGGACAAGTACAAGTCTATCAATGACGTGAACGATCTGGTGGAGAAGTATATCGATGAGAAGGAGAGGAATGAGTTCAAGAACAGCAATGCCAAGCAGTACATCCGCGAGATCTCCAACATCATCACCGACACAGAGACAGCCCACCTGGAGTATGACGATCACATCTCTCTGATCGAGAGCGAGGAGAAGGCCGACGAGATGAAGAAGCGGCTGGATATGTATATGAACATGTACCACTGGGCCAAGGCCTTTATCGTGGACGAGGTGCTGGACAGAGATGAGATGTTCTACAGCGATATCGACGATATCTATAATATCCTGGAGAACATCGTGCCACTGTATAATCGGGTGAGAAACTACGTGACCCAGAAGCCCTACAACTCTAAGAAGATCAAGCTGAATTTCCAGAGCCCTACACTGGCCAATGGCTGGTCCCAGTCTAAGGAGTTCGACAACAATGCCATCATCCTGATCAGAGATAACAAGTACTATCTGGCCATCTTCAATGCCAAGAACAAGCCAGACAAGAAGATCATCCAGGGCAACTCCGATAAGAAGAACGACAACGATTACAAGAAGATGGTGTATAACCTGCTGCCAGGCGCCAACAAGATGCTGCCCAAGGTGTTTCTGTCTAAGAAGGGCATCGAGACATTCAAGCCCTCCGACTATATCATCTCTGGCTACAACGCCCACAAGCACATCAAGACAAGCGAGAATTTTGATATCTCCTTCTGTCGGGACCTGATCGATTACTTCAAGAACAGCATCGAGAAGCACGCCGAGTGGAGAAAGTATGAGTTCAAGTTTTCCGCCACCGACAGCTACTCCGATATCTCTGAGTTCTATCGGGAGGTGGAGATGCAGGGCTACAGAATCGACTGGACATATATCAGCGAGGCCGACATCAACAAGCTGGATGAGGAGGGCAAGATCTATCTGTTTCAGATCTACAATAAGGATTTCGCCGAGAACAGCACCGGCAAGGAGAATCTGCACACAATGTACTTTAAGAACATCTTCTCCGAGGAGAATCTGAAGGACATCATCATCAAGCTGAACGGCCAGGCCGAGCTGTTTTATCGGAGAGCCTCTGTGAAGAATCCCGTGAAGCACAAGAAGGATAGCGTGCTGGTGAACAAGACCTACAAGAATCAGCTGGACAACGGCGACGTGGTGAGAATCCCCATCCCTGACGATATCTATAACGAGATCTACAAGATGTATAATGGCTACATCAAGGAGTCCGACCTGTCTGAGGCCGCCAAGGAGTACCTGGATAAGGTGGAGGTGAGGACCGCCCAGAAGGACATCGTGAAGGATTACCGCTATACAGTGGACAAGTACTTCATCCACACACCTATCACCATCAACTATAAGGTGACCGCCCGCAACAATGTGAATGATATGGTGGTGAAGTACATCGCCCAGAACGACGATATCCACGTGATCGGCATCGACCGGGGCGAGAGAAACCTGATCTACATCTCCGTGATCGATTCTCACGGCAACATCGTGAAGCAGAAATCCTACAACATCCTGAACAACTACGACTACAAGAAGAAGCTGGTGGAGAAGGAGAAAACCCGGGAGTACGCCAGAAAGAACTGGAAGAGCATCGGCAATATCAAGGAGCTGAAGGAGGGCTATATCTCCGGCGTGGTGCACGAGATCGCCATGCTGATCGTGGAGTACAACGCCATCATCGCCATGGAGGACCTGAATTATGGCTTTAAGAGGGGCCGCTTCAAGGTGGAGCGGCAGGTGTACCAGAAGTTTGAGAGCATGCTGATCAATAAGCTGAACTATTTCGCCAGCAAGGAGAAGTCCGTGGACGAGCCAGGAGGCCTGCTGAAGGGCTATCAGCTGACCTACGTGCCCGATAATATCAAGAACCTGGGCAAGCAGTGCGGCGTGATCTTTTACGTGCCTGCCGCCTTCACCAGCAAGATCGACCCATCCACAGGCTTTATCTCTGCCTTCAACTTTAAGTCTATCAGCACAAATGCCTCTCGGAAGCAGTTCTTTATGCAGTTTGACGAGATCAGATACTGTGCCGAGAAGGATATGTTCAGCTTTGGCTTCGACTACAACAACTTCGATACCTACAACATCACAATGGGCAAGACACAGTGGACCGTGTATACAAACGGCGAGAGACTGCAGTCTGAGTTCAACAATGCCAGGCGCACCGGCAAGACAAAGAGCATCAATCTGACAGAGACAATCAAGCTGCTGCTGGAGGACAATGAGATCAACTACGCCGACGGCCACGATATCAGGATCGATATGGAGAAGATGGACGAGGATAAGAAGAGCGAGTTCTTTGCCCAGCTGCTGAGCCTGTATAAGCTGACCGTGCAGATGCGCAATTCCTATACAGAGGCCGAGGAGCAGGAGAACGGCATCTCTTACGACAAGATCATCAGCCCTGTGATCAATGATGAGGGCGAGTTCTTTGACTCCGATAACTATAAGGAGTCTGACGATAAGGAGTGCAAGATGCCAAAGGACGCCGATGCCAACGGCGCCTACTGTATCGCCCTGAAGGGCCTGTATGAGGTGCTGAAGATCAAGAGCGAGTGGACCGAGGACGGCTTTGATAGGAATTGCCTGAAGCTGCCACACGCAGAGTGGCTGGACTTCATCCAGAACAAGCGGTACGAGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGC CTAAGAATTC12- Moraxella bovoculi 237 (MbCpfl) (SEQ ID NO: 224)ATGCTGTTCCAGGACTTTACCCACCTGTATCCACTGTCCAAGACAGTGAGATTTGAGCTGAAGCCCATCGATAGGACCCTGGAGCACATCCACGCCAAGAACTTCCTGTCTCAGGACGAGACAATGGCCGATATGCACCAGAAGGTGAAAGTGATCCTGGACGATTACCACCGCGACTTCATCGCCGATATGATGGGCGAGGTGAAGCTGACCAAGCTGGCCGAGTTCTATGACGTGTACCTGAAGTTTCGGAAGAACCCAAAGGACGATGAGCTGCAGAAGCAGCTGAAGGATCTGCAGGCCGTGCTGAGAAAGGAGATCGTGAAGCCCATCGGCAATGGCGGCAAGTATAAGGCCGGCTACGACAGGCTGTTCGGCGCCAAGCTGTTTAAGGACGGCAAGGAGCTGGGCGATCTGGCCAAGTTCGTGATCGCACAGGAGGGAGAGAGCTCCCCAAAGCTGGCCCACCTGGCCCACTTCGAGAAGTTTTCCACCTATTTCACAGGCTTTCACGATAACCGGAAGAATATGTATTCTGACGAGGATAAGCACACCGCCATCGCCTACCGCCTGATCCACGAGAACCTGCCCCGGTTTATCGACAATCTGCAGATCCTGACCACAATCAAGCAGAAGCACTCTGCCCTGTACGATCAGATCATCAACGAGCTGACCGCCAGCGGCCTGGACGTGTCTCTGGCCAGCCACCTGGATGGCTATCACAAGCTGCTGACACAGGAGGGCATCACCGCCTACAATACACTGCTGGGAGGAATCTCCGGAGAGGCAGGCTCTCCTAAGATCCAGGGCATCAACGAGCTGATCAATTCTCACCACAACCAGCACTGCCACAAGAGCGAGAGAATCGCCAAGCTGAGGCCACTGCACAAGCAGATCCTGTCCGACGGCATGAGCGTGTCCTTCCTGCCCTCTAAGTTTGCCGACGATAGCGAGATGTGCCAGGCCGTGAACGAGTTCTATCGCCACTACGCCGACGTGTTCGCCAAGGTGCAGAGCCTGTTCGACGGCTTTGACGATCACCAGAAGGATGGCATCTACGTGGAGCACAAGAACCTGAATGAGCTGTCCAAGCAGGCCTTCGGCGACTTTGCACTGCTGGGACGCGTGCTGGACGGATACTATGTGGATGTGGTGAATCCAGAGTTCAACGAGCGGTTTGCCAAGGCCAAGACCGACAATGCCAAGGCCAAGCTGACAAAGGAGAAGGATAAGTTCATCAAGGGCGTGCACTCCCTGGCCTCTCTGGAGCAGGCCATCGAGCACTATACCGCAAGGCACGACGATGAGAGCGTGCAGGCAGGCAAGCTGGGACAGTACTTCAAGCACGGCCTGGCCGGAGTGGACAACCCCATCCAGAAGATCCACAACAATCACAGCACCATCAAGGGCTTTCTGGAGAGGGAGCGCCCTGCAGGAGAGAGAGCCCTGCCAAAGATCAAGTCCGGCAAGAATCCTGAGATGACACAGCTGAGGCAGCTGAAGGAGCTGCTGGATAACGCCCTGAATGTGGCCCACTTCGCCAAGCTGCTGACCACAAAGACCACACTGGACAATCAGGATGGCAACTTCTATGGCGAGTTTGGCGTGCTGTACGACGAGCTGGCCAAGATCCCCACCCTGTATAACAAGGTGAGAGATTACCTGAGCCAGAAGCCTTTCTCCACCGAGAAGTACAAGCTGAACTTTGGCAATCCAACACTGCTGAATGGCTGGGACCTGAACAAGGAGAAGGATAATTTCGGCGTGATCCTGCAGAAGGACGGCTGCTACTATCTGGCCCTGCTGGACAAGGCCCACAAGAAGGTGTTTGATAACGCCCCTAATACAGGCAAGAGCATCTATCAGAAGATGATCTATAAGTACCTGGAGGTGAGGAAGCAGTTCCCCAAGGTGTTCTTTTCCAAGGAGGCCATCGCCATCAACTACCACCCTTCTAAGGAGCTGGTGGAGATCAAGGACAAGGGCCGGCAGAGATCCGACGATGAGCGCCTGAAGCTGTATCGGTTTATCCTGGAGTGTCTGAAGATCCACCCTAAGTACGATAAGAAGTTCGAGGGCGCCATCGGCGACATCCAGCTGTTTAAGAAGGATAAGAAGGGCAGAGAGGTGCCAATCAGCGAGAAGGACCTGTTCGATAAGATCAACGGCATCTTTTCTAGCAAGCCTAAGCTGGAGATGGAGGACTTCTTTATCGGCGAGTTCAAGAGGTATAACCCAAGCCAGGACCTGGTGGATCAGTATAATATCTACAAGAAGATCGACTCCAACGATAATCGCAAGAAGGAGAATTTCTACAACAATCACCCCAAGTTTAAGAAGGATCTGGTGCGGTACTATTACGAGTCTATGTGCAAGCACGAGGAGTGGGAGGAGAGCTTCGAGTTTTCCAAGAAGCTGCAGGACATCGGCTGTTACGTGGATGTGAACGAGCTGTTTACCGAGATCGAGACACGGAGACTGAATTATAAGATCTCCTTCTGCAACATCAATGCCGACTACATCGATGAGCTGGTGGAGCAGGGCCAGCTGTATCTGTTCCAGATCTACAACAAGGACTTTTCCCCAAAGGCCCACGGCAAGCCCAATCTGCACACCCTGTACTTCAAGGCCCTGTTTTCTGAGGACAACCTGGCCGATCCTATCTATAAGCTGAATGGCGAGGCCCAGATCTTCTACAGAAAGGCCTCCCTGGACATGAACGAGACAACAATCCACAGGGCCGGCGAGGTGCTGGAGAACAAGAATCCCGATAATCCTAAGAAGAGACAGTTCGTGTACGACATCATCAAGGATAAGAGGTACACACAGGACAAGTTCATGCTGCACGTGCCAATCACCATGAACTTTGGCGTGCAGGGCATGACAATCAAGGAGTTCAATAAGAAGGTGAACCAGTCTATCCAGCAGTATGACGAGGTGAACGTGATCGGCATCGATCGGGGCGAGAGACACCTGCTGTACCTGACCGTGATCAATAGCAAGGGCGAGATCCTGGAGCAGTGTTCCCTGAACGACATCACCACAGCCTCTGCCAATGGCACACAGATGACCACACCTTACCACAAGATCCTGGATAAGAGGGAGATCGAGCGCCTGAACGCCCGGGTGGGATGGGGCGAGATCGAGACAATCAAGGAGCTGAAGTCTGGCTATCTGAGCCACGTGGTGCACCAGATCAGCCAGCTGATGCTGAAGTACAACGCCATCGTGGTGCTGGAGGACCTGAATTTCGGCTTTAAGAGGGGCCGCTTTAAGGTGGAGAAGCAGATCTATCAGAACTTCGAGAATGCCCTGATCAAGAAGCTGAACCACCTGGTGCTGAAGGACAAGGCCGACGATGAGATCGGCTCTTACAAGAATGCCCTGCAGCTGACCAACAATTTCACAGATCTGAAGAGCATCGGCAAGCAGACCGGCTTCCTGTTTTATGTGCCCGCCTGGAACACCTCTAAGATCGACCCTGAGACAGGCTTTGTGGATCTGCTGAAGCCAAGATACGAGAACATCGCCCAGAGCCAGGCCTTCTTTGGCAAGTTCGACAAGATCTGCTATAATGCCGACAAGGATTACTTCGAGTTTCACATCGACTACGCCAAGTTTACCGATAAGGCCAAGAATAGCCGCCAGATCTGGACAATCTGTTCCCACGGCGACAAGCGGTACGTGTACGATAAGACAGCCAACCAGAATAAGGGCGCCGCCAAGGGCATCAACGTGAATGATGAGCTGAAGTCCCTGTTCGCCCGCCACCACATCAACGAGAAGCAGCCCAACCTGGTCATGGACATCTGCCAGAACAATGATAAGGAGTTTCACAAGTCTCTGATGTACCTGCTGAAAACCCTGCTGGCCCTGCGGTACAGCAACGCCTCCTCTGACGAGGATTTCATCCTGTCCCCCGTGGCAAACGACGAGGGCGTGTTCTTTAATAGCGCCCTGGCCGACGATACACAGCCTCAGAATGCCGATGCCAACGGCGCCTACCACATCGCCCTGAAGGGCCTGTGGCTGCTGAATGAGCTGAAGAACTCCGACGATCTGAACAAGGTGAAGCTGGCCATCGACAATCAGACCTGGCTGAATTTCGCCCAGAACAGGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTC 13- Leptospira inadai (LiCpfl)(SEQ ID NO: 225) ATGGAGGACTATTCCGGCTTTGTGAACATCTACTCTATCCAGAAAACCCTGAGGTTCGAGCTGAAGCCAGTGGGCAAGACACTGGAGCACATCGAGAAGAAGGGCTTCCTGAAGAAGGACAAGATCCGGGCCGAGGATTACAAGGCCGTGAAGAAGATCATCGATAAGTACCACAGAGCCTATATCGAGGAGGTGTTTGATTCCGTGCTGCACCAGAAGAAGAAGAAGGACAAGACCCGCTTTTCTACACAGTTCATCAAGGAGATCAAGGAGTTCAGCGAGCTGTACTATAAGACCGAGAAGAACATCCCCGACAAGGAGAGGCTGGAGGCCCTGAGCGAGAAGCTGCGCAAGATGCTGGTGGGCGCCTTTAAGGGCGAGTTCTCCGAGGAGGTGGCCGAGAAGTATAAGAACCTGTTTTCTAAGGAGCTGATCAGGAATGAGATCGAGAAGTTCTGCGAGACAGACGAGGAGCGCAAGCAGGTGTCTAACTTCAAGAGCTTCACCACATACTTTACCGGCTTCCACTCCAACAGGCAGAATATCTATTCCGACGAGAAGAAGTCTACAGCCATCGGCTACCGCATCATCCACCAGAACCTGCCTAAGTTCCTGGATAATCTGAAGATCATCGAGTCCATCCAGCGGCGGTTCAAGGACTTCCCATGGTCTGATCTGAAGAAGAACCTGAAGAAGATCGATAAGAATATCAAGCTGACCGAGTACTTCAGCATCGACGGCTTCGTGAACGTGCTGAATCAGAAGGGCATCGATGCCTACAACACAATCCTGGGCGGCAAGTCCGAGGAGTCTGGCGAGAAGATCCAGGGCCTGAACGAGTACATCAATCTGTATCGGCAGAAGAACAATATCGACAGAAAGAACCTGCCCAATGTGAAGATCCTGTTTAAGCAGATCCTGGGCGATAGGGAGACAAAGAGCTTTATCCCTGAGGCCTTCCCAGACGATCAGTCCGTGCTGAACTCTATCACAGAGTTCGCCAAGTACCTGAAGCTGGATAAGAAGAAGAAGAGCATCATCGCCGAGCTGAAGAAGTTTCTGAGCTCCTTCAATCGCTACGAGCTGGACGGCATCTATCTGGCCAACGATAATAGCCTGGCCTCTATCAGCACCTTCCTGTTTGACGATTGGTCCTTTATCAAGAAGTCCGTGTCTTTCAAGTATGACGAGTCCGTGGGCGACCCCAAGAAGAAGATCAAGTCTCCCCTGAAGTACGAGAAGGAGAAGGAGAAGTGGCTGAAGCAGAAGTACTATACAATCTCTTTCCTGAACGATGCCATCGAGAGCTATTCCAAGTCTCAGGACGAGAAGAGGGTGAAGATCCGCCTGGAGGCCTACTTTGCCGAGTTCAAGAGCAAGGACGATGCCAAGAAGCAGTTCGACCTGCTGGAGAGGATCGAGGAGGCCTATGCCATCGTGGAGCCTCTGCTGGGAGCAGAGTACCCAAGGGACCGCAACCTGAAGGCCGATAAGAAGGAAGTGGGCAAGATCAAGGACTTCCTGGATAGCATCAAGTCCCTGCAGTTCTTTCTGAAGCCTCTGCTGTCCGCCGAGATCTTTGACGAGAAGGATCTGGGCTTCTACAATCAGCTGGAGGGCTACTATGAGGAGATCGATTCTATCGGCCACCTGTATAACAAGGTGCGGAATTATCTGACCGGCAAGATCTACAGCAAGGAGAAGTTTAAGCTGAACTTCGAGAACAGCACCCTGCTGAAGGGCTGGGACGAGAACCGGGAGGTGGCCAATCTGTGCGTGATCTTCAGAGAGGACCAGAAGTACTATCTGGGCGTGATGGATAAGGAGAACAATACCATCCTGTCCGACATCCCCAAGGTGAAGCCTAACGAGCTGTTTTACGAGAAGATGGTGTATAAGCTGATCCCCACACCTCACATGCAGCTGCCCCGGATCATCTTCTCTAGCGACAACCTGTCTATCTATAATCCTAGCAAGTCCATCCTGAAGATCAGAGAGGCCAAGAGCTTTAAGGAGGGCAAGAACTTCAAGCTGAAGGACTGTCACAAGTTTATCGATTTCTACAAGGAGTCTATCAGCAAGAATGAGGACTGGAGCAGATTCGACTTCAAGTTCAGCAAGACCAGCAGCTACGAGAACATCAGCGAGTTTTACCGGGAGGTGGAGAGACAGGGCTATAACCTGGACTTCAAGAAGGTGTCTAAGTTCTACATCGACAGCCTGGTGGAGGATGGCAAGCTGTACCTGTTCCAGATCTATAACAAGGACTTTTCTATCTTCAGCAAGGGCAAGCCCAATCTGCACACCATCTATTTTCGGTCCCTGTTCTCTAAGGAGAACCTGAAGGACGTGTGCCTGAAGCTGAATGGCGAGGCCGAGATGTTCTTTCGGAAGAAGTCCATCAACTACGATGAGAAGAAGAAGCGGGAGGGCCACCACCCCGAGCTGTTTGAGAAGCTGAAGTATCCTATCCTGAAGGACAAGAGATACAGCGAGGATAAGTTTCAGTTCCACCTGCCCATCAGCCTGAACTTCAAGTCCAAGGAGCGGCTGAACTTTAATCTGAAAGTGAATGAGTTCCTGAAGAGAAACAAGGACATCAATATCATCGGCATCGATCGGGGCGAGAGAAACCTGCTGTACCTGGTCATGATCAATCAGAAGGGCGAGATCCTGAAGCAGACCCTGCTGGACAGCATGCAGTCCGGCAAGGGCCGGCCTGAGATCAACTACAAGGAGAAGCTGCAGGAGAAGGAGATCGAGAGGGATAAGGCCCGCAAGAGCTGGGGCACAGTGGAGAATATCAAGGAGCTGAAGGAGGGCTATCTGTCTATCGTGATCCACCAGATCAGCAAGCTGATGGTGGAGAACAATGCCATCGTGGTGCTGGAGGACCTGAACATCGGCTTTAAGCGGGGCAGACAGAAGGTGGAGCGGCAGGTGTACCAGAAGTTCGAGAAGATGCTGATCGATAAGCTGAACTTTCTGGTGTTCAAGGAGAATAAGCCAACCGAGCCAGGAGGCGTGCTGAAGGCCTATCAGCTGACAGACGAGTTTCAGTCTTTCGAGAAGCTGAGCAAGCAGACCGGCTTTCTGTTCTACGTGCCAAGCTGGAACACCTCCAAGATCGACCCCAGAACAGGCTTTATCGATTTCCTGCACCCTGCCTACGAGAATATCGAGAAGGCCAAGCAGTGGATCAACAAGTTTGATTCCATCAGGTTCAATTCTAAGATGGACTGGTTTGAGTTCACCGCCGATACACGCAAGTTTTCCGAGAACCTGATGCTGGGCAAGAATCGGGTGTGGGTCATCTGCACCACAAATGTGGAGCGGTACTTCACCAGCAAGACCGCCAACAGCTCCATCCAGTACAATAGCATCCAGATCACCGAGAAGCTGAAGGAGCTGTTTGTGGACATCCCTTTCAGCAACGGCCAGGATCTGAAGCCAGAGATCCTGAGGAAGAATGACGCCGTGTTCTTTAAGAGCCTGCTGTTTTACATCAAGACCACACTGTCCCTGCGCCAGAACAATGGCAAGAAGGGCGAGGAGGAGAAGGACTTCATCCTGAGCCCAGTGGTGGATTCCAAGGGCCGGTTCTTTAACTCTCTGGAGGCCAGCGACGATGAGCCCAAGGACGCCGATGCCAATGGCGCCTACCACATCGCCCTGAAGGGCCTGATGAACCTGCTGGTGCTGAATGAGACAAAGGAGGAGAACCTGAGCAGACCAAAGTGGAAGATCAAGAATAAGGACTGGCTGGAGTTCGTGTGGGAGAGGAACCGCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTC 14- Lachnospiraceae bacterium ND2006 (LbCpfl)(SEQ ID NO: 226) ATGAGCAAGCTGGAGAAGTTTACAAACTGCTACTCCCTGTCTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGACCCAGGAGAACATCGACAATAAGCGGCTGCTGGTGGAGGACGAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGCTGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCACAGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTGTTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGCTGGAGAACCTGGAGATCAATCTGCGGAAGGAGATCGCCAAGGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAGAAGGATATCATCGAGACAATCCTGCCAGAGTTCCTGGACGATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTTTACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAATATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCAGGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATATGGACATCTTCGAGAAGGTGGACGCCATCTTTGATAAGCACGAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACTATGATGTGGAGGATTTCTTTGAGGGCGAGTTCTTTAACTTTGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATCATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGGGCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAAGCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTGCTGAGCGATCGGGAGTCTCTGAGCTTCTACGGCGAGGGCTATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACACCCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAGCTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCGCCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAATCTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGACAAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGAAGGCCGTGGTGACCGAGAAGTACGAGGACGATCGGAGAAAGTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTGCAGGAGTACGCCGACGCCGATCTGTCTGTGGTGGAGAAGCTGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAAGGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATTTTGTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGGCCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGAGAATTACATCAAGGCCTTCTTTGGCGAGGGCAAGGAGACAAACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCTACGACATCCTGCTGAAGGTGGACCACATCTACGATGCCATCCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAGTTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGGCTGGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCTGAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAGAAGTACGCCAAGTGCCTGCAGAAGATCGACAAGGACGATGTGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCCCGGCCCTAATAAGATGCTGCCAAAGGTGTTCTTTTCTAAGAAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGAAGATCTACAAGAATGGCACATTCAAGAAGGGCGATATGTTTAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAGGATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACGATTTCAACTTTTCTGAGACAGAGAAGTATAAGGACATCGCCGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTGAGCTTCGAGTCTGCCAGCAAGAAGGAGGTGGATAAGCTGGTGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAAGGACTTTTCCGATAAGTCTCACGGCACACCCAATCTGCACACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACGGACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAGGCGCGCCTCCCTGAAGAAGGAGGAGCTGGTGGTGCACCCAGCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCAAGAAAACCACAACCCTGTCCTACGACGTGTATAAGGATAAGAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATCGCCATCAATAAGTGCCCCAAGAACATCTTCAAGATCAATACAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTATGTGATCGGCATCGATAGGGGCGAGCGCAATCTGCTGTATATCGTGGTGGTGGACGGCAAGGGCAACATCGTGGAGCAGTATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAGGATCAAGACAGATTACCACTCTCTGCTGGACAAGAAGGAGAAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCGAGAATATCAAGGAGCTGAAGGCCGGCTATATCTCTCAGGTGGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCCGTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATAGCCGCGTGAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGAGAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAGAAGTCTAATCCTTGTGCAACAGGCGGCGCCCTGAAGGGCTATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTCTACCCAGAACGGCTTCATCTTTTACATCCCTGCCTGGCTGACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGCTGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTTCATCAGCTCCTTTGACAGGATCATGTACGTGCCCGAGGAGGATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTCGCACAGACGCCGATTACATCAAGAAGTGGAAGCTGTACTCCTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAGAACAACGTGTTCGACTGGGAGGAGGTGTGCCTGACCAGCGCCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCAGCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGACAAGGCCTTCTACTCTAGCTTTATGGCCCTGATGAGCCTGATGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGTGGATTTTCTGATCAGCCCTGTGAAGAACTCCGACGGCATCTTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCATCCTGCCAAAGAACGCCGACGCCAATGGCGCCTATAACATCGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAGGCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCTCTAACAAGGAGTGGCTGGAGTACGCCCAGACCAGCGTGAAGCACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTC 15- Porphyromonas crevioricanis (PcCpfl)(SEQ ID NO: 227) ATGGACAGCCTGAAGGATTTCACCAACCTGTACCCCGTGTCCAAGACACTGCGGTTTGAGCTGAAGCCTGTGGGCAAGACCCTGGAGAATATCGAGAAGGCCGGCATCCTGAAGGAGGATGAGCACAGAGCCGAGAGCTACCGGAGAGTGAAGAAGATCATCGATACATATCACAAGGTGTTCATCGACAGCTCCCTGGAGAACATGGCCAAGATGGGCATCGAGAATGAGATCAAGGCCATGCTGCAGTCCTTTTGCGAGCTGTATAAGAAGGACCACAGGACCGAGGGAGAGGACAAGGCCCTGGATAAGATCAGGGCCGTGCTGAGGGGCCTGATCGTGGGAGCCTTCACCGGCGTGTGCGGCCGGCGGGAGAACACAGTGCAGAATGAGAAGTATGAGAGCCTGTTTAAGGAGAAGCTGATCAAGGAGATCCTGCCAGATTTCGTGCTGTCTACAGAGGCCGAGTCCCTGCCCTTTTCTGTGGAGGAGGCCACCAGAAGCCTGAAGGAGTTCGACTCCTTTACATCTTACTTCGCCGGCTTTTATGAGAACCGGAAGAATATCTACTCTACCAAGCCCCAGAGCACAGCCATCGCCTATAGACTGATCCACGAGAACCTGCCTAAGTTCATCGATAATATCCTGGTGTTTCAGAAGATCAAGGAGCCAATCGCCAAGGAGCTGGAGCACATCAGGGCAGACTTCAGCGCCGGCGGCTACATCAAGAAGGATGAGCGCCTGGAGGACATCTTTTCCCTGAACTACTATATCCACGTGCTGTCTCAGGCCGGCATCGAGAAGTACAATGCCCTGATCGGCAAGATCGTGACCGAGGGCGATGGCGAGATGAAGGGCCTGAACGAGCACATCAACCTGTATAATCAGCAGAGGGGCCGCGAGGACCGGCTGCCACTGTTCAGACCCCTGTATAAGCAGATCCTGTCTGATAGGGAGCAGCTGTCCTATCTGCCAGAGTCTTTCGAGAAGGACGAGGAGCTGCTGAGGGCCCTGAAGGAGTTTTACGATCACATCGCAGAGGACATCCTGGGAAGGACCCAGCAGCTGATGACAAGCATCTCCGAGTACGATCTGTCCCGGATCTATGTGAGAAACGATAGCCAGCTGACCGACATCTCCAAGAAGATGCTGGGCGATTGGAATGCCATCTACATGGCCCGGGAGAGAGCCTATGACCACGAGCAGGCCCCCAAGCGCATCACAGCCAAGTACGAGAGGGACCGCATCAAGGCCCTGAAGGGCGAGGAGTCTATCAGCCTGGCCAACCTGAACAGCTGCATCGCCTTCCTGGACAACGTGAGGGATTGTCGCGTGGACACCTATCTGTCTACACTGGGACAGAAGGAGGGACCTCACGGCCTGAGCAACCTGGTGGAGAACGTGTTCGCCTCCTACCACGAGGCCGAGCAGCTGCTGTCTTTTCCCTATCCTGAGGAGAACAATCTGATCCAGGACAAGGATAACGTGGTGCTGATCAAGAACCTGCTGGATAATATCAGCGACCTGCAGAGGTTCCTGAAGCCACTGTGGGGCATGGGCGATGAGCCCGACAAGGATGAGAGGTTTTACGGCGAGTACAATTATATCAGGGGCGCCCTGGACCAGGTCATCCCTCTGTATAACAAGGTGCGGAATTATCTGACCCGCAAGCCATACTCCACACGCAAGGTGAAGCTGAACTTCGGCAATAGCCAGCTGCTGTCCGGCTGGGATAGGAACAAGGAGAAGGACAATTCTTGCGTGATCCTGCGCAAGGGCCAGAACTTCTACCTGGCCATCATGAACAATCGGCACAAGCGGAGCTTCGAGAATAAGATGCTGCCCGAGTATAAGGAGGGCGAGCCTTACTTCGAGAAGATGGATTATAAGTTTCTGCCAGACCCCAACAAGATGCTGCCCAAGGTGTTCCTGTCTAAGAAGGGCATCGAGATCTACAAGCCTAGCCCAAAGCTGCTGGAGCAGTATGGCCACGGCACCCACAAGAAGGGCGATACCTTCAGCATGGACGATCTGCACGAGCTGATCGACTTCTTTAAGCACTCCATCGAGGCCCACGAGGATTGGAAGCAGTTCGGCTTTAAGTTCAGCGACACCGCCACATACGAGAACGTGAGCAGCTTCTACCGGGAGGTGGAGGACCAGGGCTACAAGCTGTCTTTTAGAAAGGTGTCCGAGTCTTACGTGTATAGCCTGATCGATCAGGGCAAGCTGTACCTGTTCCAGATCTATAACAAGGACTTTAGCCCTTGTTCCAAGGGCACCCCAAATCTGCACACACTGTACTGGCGGATGCTGTTCGATGAGAGAAACCTGGCCGACGTGATCTATAAGCTGGATGGCAAGGCCGAGATCTTCTTTCGGGAGAAGTCCCTGAAGAATGACCACCCAACCCACCCTGCAGGCAAGCCCATCAAGAAGAAGAGCCGGCAGAAGAAGGGCGAGGAGAGCCTGTTCGAGTACGATCTGGTGAAGGACCGGAGATATACCATGGATAAGTTTCAGTTCCACGTGCCAATCACAATGAACTTTAAGTGCTCTGCCGGCAGCAAGGTGAACGACATGGTGAATGCCCACATCAGGGAGGCCAAGGACATGCACGTGATCGGCATCGATAGGGGCGAGCGCAATCTGCTGTATATCTGCGTGATCGACAGCCGCGGCACCATCCTGGATCAGATCTCCCTGAACACAATCAATGACATCGATTATCACGATCTGCTGGAGTCCAGGGACAAGGATCGCCAGCAGGAGCACAGGAACTGGCAGACCATCGAGGGCATCAAGGAGCTGAAGCAGGGCTACCTGTCTCAGGCCGTGCACCGCATCGCCGAGCTGATGGTGGCCTATAAGGCCGTGGTGGCCCTGGAGGACCTGAACATGGGCTTCAAGCGGGGCAGACAGAAGGTGGAGAGCAGCGTGTACCAGCAGTTTGAGAAGCAGCTGATCGACAAGCTGAATTATCTGGTGGATAAGAAGAAGCGGCCCGAGGACATCGGAGGCCTGCTGAGAGCCTACCAGTTCACCGCCCCTTTCAAGAGCTTTAAGGAGATGGGCAAGCAGAACGGCTTTCTGTTCTATATCCCTGCCTGGAACACATCCAATATCGACCCAACCACAGGCTTCGTGAACCTGTTTCACGTGCAGTACGAGAATGTGGATAAGGCCAAGAGCTTCTTTCAGAAGTTCGACAGCATCTCCTACAACCCTAAGAAGGATTGGTTTGAGTTCGCCTTTGACTATAAGAACTTCACCAAGAAGGCCGAGGGCTCTAGGAGCATGTGGATTCTGTGCACCCACGGCTCCCGGATCAAGAACTTCAGAAATTCTCAGAAGAATGGCCAGTGGGATAGCGAGGAGTTTGCCCTGACCGAGGCCTTCAAGTCCCTGTTTGTGCGGTACGAGATCGATTATACCGCCGACCTGAAAACCGCCATCGTGGACGAGAAGCAGAAGGATTTCTTTGTGGACCTGCTGAAGCTGTTCAAGCTGACCGTGCAGATGAGAAACTCCTGGAAGGAGAAGGACCTGGATTACCTGATCTCTCCAGTGGCCGGCGCCGATGGCAGGTTCTTTGACACACGCGAGGGCAATAAGAGCCTGCCCAAGGACGCAGATGCAAACGGAGCCTATAATATCGCCCTGAAGGGCCTGTGGGCACTGAGGCAGATCAGACAGACCTCCGAGGGCGGCAAGCTGAAGCTGGCCATCTCTAACAAGGAGTGGCTGCAGTTTGTGCAGGAGAGATCCTACGAGAAGGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAAT TC16- Prevotella disiens (PdCpfl) (SEQ ID NO: 228)ATGGAGAACTATCAGGAGTTCACCAACCTGTTTCAGCTGAATAAGACACTGAGATTCGAGCTGAAGCCCATCGGCAAGACCTGCGAGCTGCTGGAGGAGGGCAAGATCTTCGCCAGCGGCTCCTTTCTGGAGAAGGACAAGGTGAGGGCCGATAACGTGAGCTACGTGAAGAAGGAGATCGACAAGAAGCACAAGATCTTTATCGAGGAGACACTGAGCTCCTTCTCTATCAGCAACGATCTGCTGAAGCAGTACTTTGACTGCTATAATGAGCTGAAGGCCTTCAAGAAGGACTGTAAGAGCGATGAGGAGGAGGTGAAGAAAACCGCCCTGCGCAACAAGTGTACCTCCATCCAGAGGGCCATGCGCGAGGCCATCTCTCAGGCCTTTCTGAAGAGCCCCCAGAAGAAGCTGCTGGCCATCAAGAACCTGATCGAGAACGTGTTCAAGGCCGACGAGAATGTGCAGCACTTCTCCGAGTTTACCAGCTATTTCTCCGGCTTTGAGACAAACAGAGAGAATTTCTACTCTGACGAGGAGAAGTCCACATCTATCGCCTATAGGCTGGTGCACGATAACCTGCCTATCTTCATCAAGAACATCTACATCTTCGAGAAGCTGAAGGAGCAGTTCGACGCCAAGACCCTGAGCGAGATCTTCGAGAACTACAAGCTGTATGTGGCCGGCTCTAGCCTGGATGAGGTGTTCTCCCTGGAGTACTTTAACAATACCCTGACACAGAAGGGCATCGACAACTATAATGCCGTGATCGGCAAGATCGTGAAGGAGGATAAGCAGGAGATCCAGGGCCTGAACGAGCACATCAACCTGTATAATCAGAAGCACAAGGACCGGAGACTGCCCTTCTTTATCTCCCTGAAGAAGCAGATCCTGTCCGATCGGGAGGCCCTGTCTTGGCTGCCTGACATGTTCAAGAATGATTCTGAAGTGATCAAGGCCCTGAAGGGCTTCTACATCGAGGACGGCTTTGAGAACAATGTGCTGACACCTCTGGCCACCCTGCTGTCCTCTCTGGATAAGTACAACCTGAATGGCATCTTTATCCGCAACAATGAGGCCCTGAGCTCCCTGTCCCAGAACGTGTATCGGAATTTTTCTATCGACGAGGCCATCGATGCCAACGCCGAGCTGCAGACCTTCAACAATTACGAGCTGATCGCCAATGCCCTGCGCGCCAAGATCAAGAAGGAGACAAAGCAGGGCCGGAAGTCTTTCGAGAAGTACGAGGAGTATATCGATAAGAAGGTGAAGGCCATCGACAGCCTGTCCATCCAGGAGATCAACGAGCTGGTGGAGAATTACGTGAGCGAGTTTAACTCTAATAGCGGCAACATGCCAAGAAAGGTGGAGGACTACTTCAGCCTGATGAGGAAGGGCGACTTCGGCTCCAACGATCTGATCGAAAATATCAAGACCAAGCTGAGCGCCGCAGAGAAGCTGCTGGGCACAAAGTACCAGGAGACAGCCAAGGACATCTTCAAGAAGGATGAGAACTCCAAGCTGATCAAGGAGCTGCTGGACGCCACCAAGCAGTTCCAGCACTTTATCAAGCCACTGCTGGGCACAGGCGAGGAGGCAGATCGGGACCTGGTGTTCTACGGCGATTTTCTGCCCCTGTATGAGAAGTTTGAGGAGCTGACCCTGCTGTATAACAAGGTGCGGAATAGACTGACACAGAAGCCCTATTCCAAGGACAAGATCCGCCTGTGCTTCAACAAGCCTAAGCTGATGACAGGCTGGGTGGATTCCAAGACCGAGAAGTCTGACAACGGCACACAGTACGGCGGCTATCTGTTTCGGAAGAAGAATGAGATCGGCGAGTACGATTATTTTCTGGGCATCTCTAGCAAGGCCCAGCTGTTCAGAAAGAACGAGGCCGTGATCGGCGACTACGAGAGGCTGGATTACTATCAGCCAAAGGCCAATACCATCTACGGCTCTGCCTATGAGGGCGAGAACAGCTACAAGGAGGACAAGAAGCGGCTGAACAAAGTGATCATCGCCTATATCGAGCAGATCAAGCAGACAAACATCAAGAAGTCTATCATCGAGTCCATCTCTAAGTATCCTAATATCAGCGACGATGACAAGGTGACCCCATCCTCTCTGCTGGAGAAGATCAAGAAGGTGTCTATCGACAGCTACAACGGCATCCTGTCCTTCAAGTCTTTTCAGAGCGTGAACAAGGAAGTGATCGATAACCTGCTGAAAACCATCAGCCCCCTGAAGAACAAGGCCGAGTTTCTGGACCTGATCAATAAGGATTATCAGATCTTCACCGAGGTGCAGGCCGTGATCGACGAGATCTGCAAGCAGAAAACCTTCATCTACTTTCCAATCTCCAACGTGGAGCTGGAGAAGGAGATGGGCGATAAGGACAAGCCCCTGTGCCTGTTCCAGATCAGCAATAAGGATCTGTCCTTCGCCAAGACCTTTAGCGCCAACCTGCGGAAGAAGAGAGGCGCCGAGAATCTGCACACAATGCTGTTTAAGGCCCTGATGGAGGGCAACCAGGATAATCTGGACCTGGGCTCTGGCGCCATCTTCTACAGAGCCAAGAGCCTGGACGGCAACAAGCCCACACACCCTGCCAATGAGGCCATCAAGTGTAGGAACGTGGCCAATAAGGATAAGGTGTCCCTGTTCACCTACGACATCTATAAGAACAGGCGCTACATGGAGAATAAGTTCCTGTTTCACCTGAGCATCGTGCAGAACTATAAGGCCGCCAATGACTCCGCCCAGCTGAACAGCTCCGCCACCGAGTATATCAGAAAGGCCGATGACCTGCACATCATCGGCATCGATAGGGGCGAGCGCAATCTGCTGTACTATTCCGTGATCGATATGAAGGGCAACATCGTGGAGCAGGACTCTCTGAATATCATCAGGAACAATGACCTGGAGACAGATTACCACGACCTGCTGGATAAGAGGGAGAAGGAGCGCAAGGCCAACCGGCAGAATTGGGAGGCCGTGGAGGGCATCAAGGACCTGAAGAAGGGCTACCTGAGCCAGGCCGTGCACCAGATCGCCCAGCTGATGCTGAAGTATAACGCCATCATCGCCCTGGAGGATCTGGGCCAGATGTTTGTGACCCGCGGCCAGAAGATCGAGAAGGCCGTGTACCAGCAGTTCGAGAAGAGCCTGGTGGATAAGCTGTCCTACCTGGTGGACAAGAAGCGGCCTTATAATGAGCTGGGCGGCATCCTGAAGGCCTACCAGCTGGCCTCTAGCATCACCAAGAACAATTCTGACAAGCAGAACGGCTTCCTGTTTTATGTGCCAGCCTGGAATACAAGCAAGATCGATCCCGTGACCGGCTTTACAGACCTGCTGCGGCCCAAGGCCATGACCATCAAGGAGGCCCAGGACTTCTTTGGCGCCTTCGATAACATCTCTTACAATGACAAGGGCTATTTCGAGTTTGAGACAAACTACGACAAGTTTAAGATCAGAATGAAGAGCGCCCAGACCAGGTGGACAATCTGCACCTTCGGCAATCGGATCAAGAGAAAGAAGGATAAGAACTACTGGAATTATGAGGAGGTGGAGCTGACCGAGGAGTTCAAGAAGCTGTTTAAGGACAGCAACATCGATTACGAGAACTGTAATCTGAAGGAGGAGATCCAGAACAAGGACAATCGCAAGTTCTTTGATGACCTGATCAAGCTGCTGCAGCTGACACTGCAGATGCGGAACTCCGATGACAAGGGCAATGATTATATCATCTCTCCTGTGGCCAACGCCGAGGGCCAGTTCTTTGACTCCCGCAATGGCGATAAGAAGCTGCCACTGGATGCAGACGCAAACGGAGCCTACAATATCGCCCGCAAGGGCCTGTGGAACATCCGGCAGATCAAGCAGACCAAGAACGACAAGAAGCTGAATCTGAGCATCTCCTCTACAGAGTGGCTGGATTTCGTGCGGGAGAAGCCTTACCTGAAGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAA G GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGA ATTC17- Porphyromonas macacae (PmCpfl) (SEQ ID NO: 229)ATGAAAACCCAGCACTTCTTTGAGGACTTCACAAGCCTGTACTCTCTGAGCAAGACCATCCGGTTTGAGCTGAAGCCAATCGGCAAGACCCTGGAGAACATCAAGAAGAATGGCCTGATCCGGAGAGATGAGCAGAGACTGGACGATTACGAGAAGCTGAAGAAAGTGATCGACGAGTATCACGAGGATTTCATCGCCAACATCCTGAGCTCCTTTTCCTTCTCTGAGGAGATCCTGCAGTCCTACATCCAGAATCTGAGCGAGTCCGAGGCCAGGGCCAAGATCGAGAAAACCATGCGCGACACACTGGCCAAGGCCTTCTCTGAGGATGAGAGGTACAAGAGCATCTTTAAGAAGGAGCTGGTGAAGAAGGACATCCCCGTGTGGTGCCCTGCCTATAAGAGCCTGTGCAAGAAGTTCGATAACTTTACCACATCTCTGGTGCCCTTCCACGAGAACAGGAAGAACCTGTATACCAGCAATGAGATCACAGCCTCTATCCCTTATCGCATCGTGCACGTGAACCTGCCAAAGTTTATCCAGAATATCGAGGCCCTGTGCGAGCTGCAGAAGAAGATGGGCGCCGACCTGTACCTGGAGATGATGGAGAACCTGCGCAACGTGTGGCCCAGCTTCGTGAAAACCCCAGACGACCTGTGCAACCTGAAAACCTATAATCACCTGATGGTGCAGTCTAGCATCAGCGAGTACAACAGGTTTGTGGGCGGCTATTCCACCGAGGACGGCACAAAGCACCAGGGCATCAACGAGTGGATCAATATCTACAGACAGAGGAATAAGGAGATGCGCCTGCCTGGCCTGGTGTTCCTGCACAAGCAGATCCTGGCCAAGGTGGACTCCTCTAGCTTCATCAGCGATACACTGGAGAACGACGATCAGGTGTTTTGCGTGCTGAGACAGTTCAGGAAGCTGTTTTGGAATACCGTGTCCTCTAAGGAGGACGATGCCGCCTCCCTGAAGGACCTGTTCTGTGGCCTGTCTGGCTATGACCCTGAGGCCATCTACGTGAGCGATGCCCACCTGGCCACAATCTCCAAGAACATCTTTGACAGATGGAATTACATCTCCGATGCCATCAGGCGCAAGACCGAGGTGCTGATGCCACGGAAGAAGGAGAGCGTGGAGAGATATGCCGAGAAGATCTCCAAGCAGATCAAGAAGAGACAGTCTTACAGCCTGGCCGAGCTGGACGATCTGCTGGCCCACTATAGCGAGGAGTCCCTGCCCGCAGGCTTCTCTCTGCTGAGCTACTTTACATCTCTGGGCGGCCAGAAGTATCTGGTGAGCGACGGCGAAGTGATCCTGTACGAGGAGGGCAGCAACATCTGGGACGAGGTGCTGATCGCCTTCAGGGATCTGCAGGTCATCCTGGACAAGGACTTCACCGAGAAGAAGCTGGGCAAGGATGAGGAGGCCGTGTCTGTGATCAAGAAGGCCCTGGACAGCGCCCTGCGCCTGCGGAAGTTCTTTGATCTGCTGTCCGGCACAGGCGCAGAGATCAGGAGAGACAGCTCCTTCTATGCCCTGTATACCGACCGGATGGATAAGCTGAAGGGCCTGCTGAAGATGTATGATAAGGTGAGAAACTACCTGACCAAGAAGCCTTATTCCATCGAGAAGTTCAAGCTGCACTTTGACAACCCATCCCTGCTGTCTGGCTGGGATAAGAATAAGGAGCTGAACAATCTGTCTGTGATCTTCCGGCAGAACGGCTACTATTACCTGGGCATCATGACACCCAAGGGCAAGAATCTGTTCAAGACCCTGCCTAAGCTGGGCGCCGAGGAGATGTTTTATGAGAAGATGGAGTACAAGCAGATCGCCGAGCCTATGCTGATGCTGCCAAAGGTGTTCTTTCCCAAGAAAACCAAGCCAGCCTTCGCCCCAGACCAGAGCGTGGTGGATATCTACAACAAGAAAACCTTCAAGACAGGCCAGAAGGGCTTTAATAAGAAGGACCTGTACCGGCTGATCGACTTCTACAAGGAGGCCCTGACAGTGCACGAGTGGAAGCTGTTTAACTTCTCCTTTTCTCCAACCGAGCAGTATCGGAATATCGGCGAGTTCTTTGACGAGGTGAGAGAGCAGGCCTACAAGGTGTCCATGGTGAACGTGCCCGCCTCTTATATCGACGAGGCCGTGGAGAACGGCAAGCTGTATCTGTTCCAGATCTACAATAAGGACTTCAGCCCCTACTCCAAGGGCATCCCTAACCTGCACACACTGTATTGGAAGGCCCTGTTCAGCGAGCAGAATCAGAGCCGGGTGTATAAGCTGTGCGGAGGAGGAGAGCTGTTTTATAGAAAGGCCAGCCTGCACATGCAGGACACCACAGTGCACCCCAAGGGCATCTCTATCCACAAGAAGAACCTGAATAAGAAGGGCGAGACAAGCCTGTTCAACTACGACCTGGTGAAGGATAAGAGGTTTACCGAGGACAAGTTCTTTTTCCACGTGCCTATCTCTATCAACTACAAGAATAAGAAGATCACCAACGTGAATCAGATGGTGCGCGATTATATCGCCCAGAACGACGATCTGCAGATCATCGGCATCGACCGCGGCGAGCGGAATCTGCTGTATATCAGCCGGATCGATACAAGGGGCAACCTGCTGGAGCAGTTCAGCCTGAATGTGATCGAGTCCGACAAGGGCGATCTGAGAACCGACTATCAGAAGATCCTGGGCGATCGCGAGCAGGAGCGGCTGAGGCGCCGGCAGGAGTGGAAGTCTATCGAGAGCATCAAGGACCTGAAGGATGGCTACATGAGCCAGGTGGTGCACAAGATCTGTAACATGGTGGTGGAGCACAAGGCCATCGTGGTGCTGGAGAACCTGAATCTGAGCTTCATGAAGGGCAGGAAGAAGGTGGAGAAGTCCGTGTACGAGAAGTTTGAGCGCATGCTGGTGGACAAGCTGAACTATCTGGTGGTGGATAAGAAGAACCTGTCCAATGAGCCAGGAGGCCTGTATGCAGCATACCAGCTGACCAATCCACTGTTCTCTTTTGAGGAGCTGCACAGATACCCCCAGAGCGGCATCCTGTTTTTCGTGGACCCATGGAACACCTCTCTGACAGATCCCAGCACAGGCTTCGTGAATCTGCTGGGCAGAATCAACTACACCAATGTGGGCGACGCCCGCAAGTTTTTCGATCGGTTTAACGCCATCAGATATGACGGCAAGGGCAATATCCTGTTCGACCTGGATCTGTCCAGATTTGATGTGAGGGTGGAGACACAGAGGAAGCTGTGGACACTGACCACATTCGGCTCTCGCATCGCCAAATCCAAGAAGTCTGGCAAGTGGATGGTGGAGCGGATCGAGAACCTGAGCCTGTGCTTTCTGGAGCTGTTCGAGCAGTTTAATATCGGCTACAGAGTGGAGAAGGACCTGAAGAAGGCCATCCTGAGCCAGGATAGGAAGGAGTTCTATGTGCGCCTGATCTACCTGTTTAACCTGATGATGCAGATCCGGAACAGCGACGGCGAGGAGGATTATATCCTGTCTCCCGCCCTGAACGAGAAGAATCTGCAGTTCGACAGCAGGCTGATCGAGGCCAAGGATCTGCCTGTGGACGCAGATGCAAACGGAGCATACAATGTGGCCCGCAAGGGCCTGATGGTGGTGCAGAGAATCAAGAGGGGCGACCACGAGTCCATCCACAGGATCGGAAGGGCACAGTGGCTGAGATATGTGCAGGAGGGCATCGTGGAGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG GGATCC TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAATTCAmino acid sequence of human codon optimized Cpfl orthologsNuclear localization signal (NLS) Glycine-Serine linker 3x HA tag1- Franscisella tularensis subsp. novicida U112 (FnCpfl)(SEQ ID NO: 230)MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGITYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNFINWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA3- Lachnospiraceae bacterium MC2017 (Lb3Cpf1) (SEQ ID NO: 231)MDYGNGQFERRAPLTKTITLRLKPIGETRETIREQKLLEQDAAFRKLVETVTPIVDDCIRKIADNALCHFGTEYDFSCLGNATSKNDSKAIKKETEKVEKLLAKVLTENLPDGLRKVNDINSAAFIQDTLTSFVQDDADKRVLIQELKGKTVLMQRFLTTRITALTVWLPDRVFENFNIFIENAEKMRILLDSPLNEKIMKFDPDAEQYASLEFYGQCLSQKDIDSYNLIISGIYADDEVKNPGINEIVKEYNQQIRGDKDESPLPKLKKLHKQILMPVEKAFFVRVLSNDSDARSILEKILKDTEMLPSKIIEAMKEADAGDIAVYGSRLHELSHVIYGDHGKLSQIIYDKESKRISELMETLSPKERKESKKRLEGLEEHIRKSTYTFDELNRYAEKNVMAAYIAAVEESCAEIMRKEKDLRTLLSKEDVKIRGNRHNTLIVKNYFNAWTVFRNLIRILRRKSEAEIDSDFYDVLDDSVEVLSLTYKGENLCRSYITKKIGSDLKPEIATYGSALRPNSRWWSPGEKFNVKFHTIVRRDGRLYYFILPKGAKPVELEDMDGDIECLQMRKIPNPTIFLPKLVFKDPEAFFRDNPEADEFVFLSGMKAPVTITRETYEAYRYKLYTVGKLRDGEVSEEEYKRALLQVLTAYKEFLENRMIYADLNFGFKDLEEYKDSSEFIKQVETHNTFMCWAKVSSSQLDDLVKSGNGLLFEIWSERLESYYKYGNEKVLRGYEGVLLSILKDENLVSMRTLLNSRPMLVYRPKESSKPMVVHRDGSRVVDRFDKDGKYIPPEVHDELYRFFNNLLIKEKLGEKARKILDNKKVKVKVLESERVKWSKFYDEQFAVTFSVKKNADCLDTTKDLNAEVMEQYSESNRLILIRNTTDILYYLVLDKNGKVLKQRSLNIINDGARDVDWKERFRQVTKDRNEGYNEWDYSRTSNDLKEVYLNYALKEIAEAVIEYNAILIIEKMSNAFKDKYSFLDDVTFKGFETKLLAKLSDLHFRGIKDGEPCSFTNPLQLCQNDNKILQDGVIFMVPNSMTRSLDPDTGFIFAINDHNIRTKKAKLNFLSKFDQLKVSSEGCLIMKYSGDSLPTHNTDNRVWNCCCNHPITNYDRETKKVEFIEEPVEELSRVLEENGIETDTELNKLNERENVPGKVVDAIYSLVLNYLRGTVSGVAGQRAVYYSPVTGKKYDISFIQAMNLNRKCDYYRIGSKERGEWTDFVAQLINKRPAATKKAGQA KKKK GSYPYDVPDYAYPYDVPDYAYPYDVPDYA 4- Butyrivibrio proteoclasticus (BpCpfl)(SEQ ID NO: 232)MLLYENYTKRNQITKSLRLELRPQGKTLRNIKELNLLEQDKAIYALLERLKPVIDEGIKDIARDTLKNCELSFEKLYEHFLSGDKKAYAKESERLKKEIVKTLIKNLPEGIGKISEINSAKYLNGVLYDFIDKTHKDSEEKQNILSDILETKGYLALFSKFLTSRITTLEQSMPKRVIENFEIYAANIPKMQDALERGAVSFAIEYESICSVDYYNQILSQEDIDSYNRLISGIMDEDGAKEKGINQTISEKNIKIKSEHLEEKPFRILKQLHKQILEEREKAFTIDHIDSDEEVVQVTKEAFEQTKEQWENIKKINGFYAKDPGDITLFIVVGPNQTHVLSQLIYGEHDRIRLLLEEYEKNTLEVLPRRTKSEKARYDKFVNAVPKKVAKESHTFDGLQKMTGDDRLFILYRDELARNYMRIKEAYGTFERDILKSRRGIKGNRDVQESLVSFYDELTKFRSALRIINSGNDEKADPIFYNTFDGIFEKANRTYKAENLCRNYVTKSPADDARIMASCLGTPARLRTHWWNGEENFAINDVAMIRRGDEYYYFVLTPDVKPVDLKTKDETDAQIFVQRKGAKSFLGLPKALFKCILEPYFESPEHKNDKNCVIEEYVSKPLTIDRRAYDIFKNGTFKKTNIGIDGLTEEKFKDDCRYLIDVYKEFIAVYTRYSCFNMSGLKRADEYNDIGEFFSDVDTRLCTMEWIPVSFERINDMVDKKEGLLFLVRSMFLYNRPRKPYERTFIQLFSDSNMEHTSMLLNSRAMIQYRAASLPRRVTHKKGSILVALRDSNGEHIPMHIREAIYKMKNNFDISSEDFIMAKAYLAEHDVAIKKANEDIIRNRRYTEDKFFLSLSYTKNADISARTLDYINDKVEEDTQDSRMAVIVTRNLKDLTYVAVVDEKNNVLEEKSLNEIDGVNYRELLKERTKIKYHDKTRLWQYDVSSKGLKEAYVELAVTQISKLATKYNAVVVVESMSSTFKDKFSFLDEQIFKAFEARLCARMSDLSFNTIKEGEAGSISNPIQVSNNNGNSYQDGVIYFLNNAYTRTLCPDTGFVDVFDKTRLITMQSKRQFFAKMKDIRIDDGEMLFTFNLEEYPTKRLLDRKEWTVKIAGDGSYFDKDKGEYVYVNDIVREQIIPALLEDKAVFDGNMAEKFLDKTAISGKSVELIYKWFANALYGIITKKDGEKIYRSPITGTEIDVSKNTTYNFGKKFMFKQEYRGDGDFLDAFLNYMQAQDIAVKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA5- Peregrinibacteria bacterium GW2011_GWA_33_10 (PeCpfl)(SEQ ID NO: 233)MSNFFKNFTNLYELSKTLRFELKPVGDTLTNMKDHLEYDEKLQTFLKDQNIDDAYQALKPQFDEIHEEFITDSLESKKAKEIDFSEYLDLFQEKKELNDSEKKLRNKIGETFNKAGEKWKKEKYPQYEWKKGSKIANGADILSCQDMLQFIKYKNPEDEKIKNYIDDTLKGFFTYFGGFNQNRANYYETKKEASTAVATRIVHENLPKFCDNVIQFKHIIKRKKDGTVEKTERKTEYLNAYQYLKNNNKITQIKDAETEKMIESTPIAEKIFDVYYFSSCLSQKQIEEYNRIIGHYNLLINLYNQAKRSEGKHLSANEKKYKDLPKFKTLYKQIGCGKKKDLFYTIKCDTEEEANKSRNEGKESHSVEEIINKAQEAINKYFKSNNDCENINTVPDFINYILTKENYEGVYWSKAAMNTISDKYFANYHDLQDRLKEAKVFQKADKKSEDDIKIPEAIELSGLFGVLDSLADWQTTLFKSSILSNEDKLKIITDSQTPSEALLKMIFNDIEKNMESFLKETNDIITLKKYKGNKEGTEKIKQWFDYTLAINRMLKYFLVKENKIKGNSLDTNISEALKTLIYSDDAEWFKWYDALRNYLTQKPQDEAKENKLKLNFDNPSLAGGWDVNKECSNFCVILKDKNEKKYLAIMKKGENTLFQKEWTEGRGKNLTKKSNPLFEINNCEILSKMEYDFWADVSKMIPKCSTQLKAVVNHFKQSDNEFIFPIGYKVTSGEKFREECKISKQDFELNNKVFNKNELSVTAMRYDLSSTQEKQYIKAFQKEYWELLFKQEKRDTKLTNNEIFNEWINFCNKKYSELLSWERKYKDALTNWINFCKYFLSKYPKTTLFNYSFKESENYNSLDEFYRDVDICSYKLNINTTINKSILDRLVEEGKLYLFEIKNQDSNDGKSIGHKNNLHTIYWNAIFENFDNRPKLNGEAEIFYRKAISKDKLGIVKGKKTKNGTEIIKNYRFSKEKFILHVPITLNFCSNNEYVNDIVNTKFYNFSNLHFLGIDRGEKHLAYYSLVNKNGEIVDQGTLNLPFTDKDGNQRSIKKEKYFYNKQEDKWEAKEVDCWNYNDLLDAMASNRDMARKNWQRIGTIKEAKNGYVSLVIRKIADLAVNNERPAFIVLEDLNTGFKRSRQKIDKSVYQKFELALAKKLNFLVDKNAKRDEIGSPTKALQLTPPVNNYGDIENKKQAGIMLYTRANYTSQTDPATGWRKTIYLKAGPEETTYKKDGKIKNKSVKDQIIETFTDIGFDGKDYYFEYDKGEFVDEKTGEIKPKKWRLYSGENGKSLDRFRGEREKDKYEWKIDKIDIVKILDDLFVNFDKNISLLKQLKEGVELTRNNEHGTGESLRFAINLIQQIRNTGNNERDNDFILSPVRDENGKHFDSREYWDKETKGEKISMPSSGDANGAFNIARKGIIMNAHILANSDSKDLSLFVSDEEWDLHLNNKTEWKKQLNIFSSRKAMAKRKKKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA6- Parcubacteria bacterium GWC2011_GWC2_44_17 (PbCpfl) (SEQ ID NO: 234)MENIFDQFIGKYSLSKTLRFELKPVGKTEDFLKINKVFEKDQTIDDSYNQAKFYFDSLHQKFIDAALASDKTSELSFQNFADVLEKQNKIILDKKREMGALRKRDKNAVGIDRLQKEINDAEDIIQKEKEKIYKDVRTLFDNEAESWKTYYQEREVDGKKITFSKADLKQKGADFLTAAGILKVLKYEFPEEKEKEFQAKNQPSLFVEEKENPGQKRYIFDSFDKFAGYLTKFQQTKKNLYAADGTSTAVATRIADNFIIFHQNTKVFRDKYKNNHTDLGFDEENIFEIERYKNCLLQREIEHIKNENSYNKIIGRINKKIKEYRDQKAKDTKLTKSDFPFFKNLDKQILGEVEKEKQLIEKTREKTEEDVLIERFKEFIENNEERFTAAKKLMNAFCNGEFESEYEGIYLKNKAINTISRRWFVSDRDFELKLPQQKSKNKSEKNEPKVKKFISIAEIKNAVEELDGDIFKAVFYDKKIIAQGGSKLEQFLVIWKYEFEYLFRDIERENGEKLLGYDSCLKIAKQLGIFPQEKEAREKATAVIKNYADAGLGIFQMMKYFSLDDKDRKNTPGQLSTNFYAEYDGYYKDFEFIKYYNEFRNFITKKPFDEDKIKLNFENGALLKGWDENKEYDFMGVILKKEGRLYLGIMHKNHRKLFQSMGNAKGDNANRYQKMIYKQIADASKDVPRLLLTSKKAMEKFKPSQEILRIKKEKTFKRESKNFSLRDLHALIEYYRNCIPQYSNWSFYDFQFQDTGKYQNIKEFTDDVQKYGYKISFRDIDDEYINQALNEGKMYLFEVVNKDIYNTKNGSKNLHTLYFEHILSAENLNDPVFKLSGMAEIFQRQPSVNEREKITTQKNQCILDKGDRAYKYRRYTEKKIMFHMSLVLNTGKGEIKQVQFNKIINQRISSSDNEMRVNVIGIDRGEKNLLYYSVVKQNGEIIEQASLNEINGVNYRDKLIEREKERLKNRQSWKPVVKIKDLKKGYISHVIHKICQLIEKYSAIVVLEDLNMRFKQIRGGIERSVYQQFEKALIDKLGYLVFKDNRDLRAPGGVLNGYQLSAPFVSFEKMRKQTGILFYTQAEYTSKTDPITGFRKNVYISNSASLDKIKEAVKKFDAIGWDGKEQSYFFKYNPYNLADEKYKNSTVSKEWAIFASAPRIRRQKGEDGYWKYDRVKVNEEFEKLLKVWNFVNPKATDIKQEIIKKEKAGDLQGEKELDGRLRNFWHSFIYLFNLVLELRNSFSLQIKIKAGEVIAVDEGVDFIASPVKPFFTTPNPYIPSNLCWLAVENADANGAYNIARKGVMILKKIREHAKKDPEFKKLPNLFISNAEWDEAARDWGKYAGTTALNLDHKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA7- Smithella sp. SC_K08D17 (SsCpfl) (SEQ ID NO: 235)MQTLFENFTNQYPVSKTLRFELIPQGKTKDFIEQKGLLKKDEDRAEKYKKVKNIIDEYHKDFIEKSLNGLKLDGLEKYKTLYLKQEKDDKDKKAFDKEKENLRKQIANAFRNNEKFKTLFAKELIKNDLMSFACEEDKKNVKEFEAFTTYFTGFHQNRANMYVADEKRTAIASRLIHENLPKFIDNIKIFEKMKKEAPELLSPFNQTLKDMKDVIKGTTLEEIFSLDYFNKTLTQSGIDIYNSVIGGRTPEEGKTKIKGLNEYINTDFNQKQTDKKKRQPKFKQLYKQILSDRQSLSFIAEAFKNDTEILEAIEKFYVNELLHFSNEGKSTNVLDAIKNAVSNLESFNLTKMYFRSGASLTDVSRKVFGEWSIINRALDNYYATTYPIKPREKSEKYEERKEKWLKQDFNVSLIQTAIDEYDNETVKGKNSGKVIADYFAKFCDDKETDLIQKVNEGYIAVKDLLNTPCPENEKLGSNKDQVKQIKAFMDSIMDIMHFVRPLSLKDTDKEKDETFYSLFTPLYDHLTQTIALYNKVRNYLTQKPYSTEKIKLNFENSTLLGGWDLNKETDNTAIILRKDNLYYLGIMDKRHNRIFRNVPKADKKDFCYEKMVYKLLPGANKMLPKVFFSQSRIQEFTPSAKLLENYANETHKKGDNFNLNHCHKLIDFFKDSINKHEDWKNFDFRFSATSTYADLSGFYHEVEHQGYKISFQSVADSFIDDLVNEGKLYLFQIYNKDFSPFSKGKPNLHTLYWKMLFDENNLKDVVYKLNGEAEVFYRKKSIAEKNTTIHKANESIINKNPDNPKATSTFNYDIVKDKRYTIDKFQFHIPITMNFKAEGIFNMNQRVNQFLKANPDINIIGIDRGERHLLYYALINQKGKILKQDTLNVIANEKQKVDYHNLLDKKEGDRATARQEWGVIETIKELKEGYLSQVIHKLTDLMIENNAIIVMEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVDKNKKANELGGLLNAFQLANKFESFQKMGKQNGFIFYVPAWNTSKTDPATGFIDFLKPRYENLNQAKDFFEKFDSIRLNSKADYFEFAFDFKNFTEKADGGRTKWTVCTTNEDRYAWNRALNNNRGSQEKYDITAELKSLFDGKVDYKSGKDLKQQIASQESADFFKALMKNLSITLSLRHNNGEKGDNEQDYILSPVADSKGRFFDSRKADDDMPKNADANGAYHIALKGLWCLEQISKTDDLKKVKLAISNKEWLEFVQTLKGKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA 8- Acidaminococcus sp. BV3L6 (AsCpfl) (SEQ ID NO: 236)MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLOMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA9- Lachnospiraceae bacterium MA2020 (Lb2Cpf1) (SEQ ID NO: 237)MYYESLTKQYPVSKTIRNELIPIGKTLDNIRQNNILESDVKRKQNYEHVKGILDEYHKQLINEALDNCTLPSLKIAAEIYLKNQKEVSDREDFNKTQDLLRKEVVEKLKAHENFTKIGKKDILDLLEKLPSISEDDYNALESFRNFYTYFTSYNKVRENLYSDKEKSSTVAYRLINENFPKFLDNVKSYRFVKTAGILADGLGEEEQDSLFIVETFNKTLTQDGIDTYNSQVGKINSSINLYNQKNQKANGFRKIPKMKMLYKQILSDREESFIDEFQSDEVLIDNVESYGSVLIESLKSSKVSAFFDALRESKGKNVYVKNDLAKTAMSNIVFENWRTFDDLLNQEYDLANENKKKDDKYFEKRQKELKKNKSYSLEHLCNLSEDSCNLIENYIHQISDDIENIIINNETFLRIVINEHDRSRKLAKNRKAVKAIKDFLDSIKVLERELKLINSSGQELEKDLIVYSAHEELLVELKQVDSLYNMTRNYLTKKPFSTEKVKLNFNRSTLLNGWDRNKETDNLGVLLLKDGKYYLGIMNTSANKAFVNPPVAKTEKVFKKVDYKLLPVPNQMLPKVFFAKSNIDFYNPSSEIYSNYKKGTHKKGNMFSLEDCHNLIDFFKESISKHEDWSKFGFKFSDTASYNDISEFYREVEKQGYKLTYTDIDETYINDLIERNELYLFQIYNKDFSMYSKGKLNLHTLYFMMLFDQRNIDDVVYKLNGEAEVFYRPASISEDELIIHKAGEEIKNKNPNRARTKETSTFSYDIVKDKRYSKDKFTLHIPITMNFGVDEVKRFNDAVNSAIRIDENVNVIGIDRGERNLLYVVVIDSKGNILEQISLNSIINKEYDIETDYHALLDEREGGRDKARKDWNTVENIRDLKAGYLSQVVNVVAKLVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVIDKSREQTSPKELGGALNALQLTSKFKSFKELGKQSGVIYYVPAYLTSKIDPTTGFANLFYMKCENVEKSKRFFDGFDFIRFNALENVFEFGFDYRSFTQRACGINSKWTVCTNGERIIKYRNPDKNNMFDEKVVVVTDEMKNLFEQYKIPYEDGRNVKDMIISNEEAEFYRRLYRLLQQTLQMRNSTSDGTRDYIISPVKNKREAYFNSELSDGSVPKDADANGAYNIARKGLWVLEQIRQKSEGEKINLAMTNAEWLEYAQTHLLKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA 10- Candidatus Methanoplasma termitum (CMtCpfl)(SEQ ID NO: 238)MNNYDEFTKLYPIQKTIRFELKPQGRTMEHLETFNFFEEDRDRAEKYKILKEAIDEYHKKFIDEHLTNMSLDWNSLKQISEKYYKSREEKDKKVFLSEQKRMRQEIVSEFKKDDRFKDLFSKKLFSELLKEEIYKKGNHQEIDALKSFDKFSGYFIGLHENRKNMYSDGDEITAISNRIVNENFPKFLDNLQKYQEARKKYPEWIIKAESALVAHNIKMDEVFSLEYFNKVLNQEGIQRYNLALGGYVTKSGEKMMGLNDALNLAHQSEKSSKGRIHMTPLFKQILSEKESFSYIPDVFTEDSQLLPSIGGFFAQIENDKDGNIFDRALELISSYAEYDTERIYIRQADINRVSNVIFGEWGTLGGLMREYKADSINDINLERTCKKVDKWLDSKEFALSDVLEAIKRTGNNDAFNEYISKMRTAREKIDAARKEMKFISEKISGDEESIHIIKTLLDSVQQFLHFFNLFKARQDIPLDGAFYAEFDEVHSKLFAIVPLYNKVRNYLTKNNLNTKKIKLNFKNPTLANGWDQNKVYDYASLIFLRDGNYYLGIINPKRKKNIKFEQGSGNGPFYRKMVYKQIPGPNKNLPRVFLTSTKGKKEYKPSKEIIEGYEADKHIRGDKFDLDFCHKLIDFFKESIEKHKDWSKFNFYFSPTESYGDISEFYLDVEKQGYRMHFENISAETIDEYVEKGDLFLFQIYNKDFVKAATGKKDMHTIYWNAAFSPENLQDVVVKLNGEAELFYRDKSDIKEIVHREGEILVNRTYNGRTPVPDKIHKKLTDYHNGRTKDLGEAKEYLDKVRYFKAHYDITKDRRYLNDKIYFHVPLTLNFKANGKKNLNKMVIEKFLSDEKAHIIGIDRGERNLLYYSIIDRSGKIIDQQSLNVIDGFDYREKLNQREIEMKDARQSWNAIGKIKDLKEGYLSKAVHEITKMAIQYNAIVVMEELNYGFKRGRFKVEKQIYQKFENMLIDKMNYLVFKDAPDESPGGVLNAYQLTNPLESFAKLGKQTGILFYVPAAYTSKIDPTTGFVNLFNTSSKTNAQERKEFLQKFESISYSAKDGGIFAFAFDYRKFGTSKTDHKNVWTAYTNGERMRYIKEKKRNELFDPSKEIKEALTSSGIKYDGGQNILPDILRSNNNGLIYTMYSSFIAAIQMRVYDGKEDYIISPIKNSKGEFFRTDPKRRELPIDADANGAYNIALRGELTMRAIAEKFDPDSEKMAKLELKHKDWFEFMQTRGDKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA11- Eubacterium eligens (EeCpfl) (SEQ ID NO: 239)MNGNRSIVYREFVGVIPVAKTLRNELRPVGHTQEHIIQNGLIQEDELRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSPSKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLLKEILPDFIKNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVSTSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNNYNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDDHISLIESEEKADEMKKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTENFDISFCRDLIDYFKNSIEKHAEWRKYEFKSATDSYSDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFSEENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRTAQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMVVKYIAQNDDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLIVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKEKSVDEPGGLLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYADGHDIRIDMEKMDEDKKSEFFAQLLSLYKLTVQMRNSYTEAEEQENGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYEKRPAATKKAGQA KKKK GSYPYDVPDYAYPYDVPDYAYPYDVPDYA 12- Moraxella bovoculi 237 (MbCpfl)(SEQ ID NO: 240)MLFQDFTHLYPLSKTVRFELKPIDRTLEHIHAKNFLSQDETMADMHQKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDDELQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDGKELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYSDEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTASGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSPKIQGINELINSHHNQHCHKSERIAKLRPLHKQILSDGMSVSFLPSKFADDSEMCQAVNEFYRHYADVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKSIYQKMIYKYLEVRKQFPKVFFSKEAIAINYHPSKELVEIKDKGRQRSDDERLKLYRFILECLKIHPKYDKKFEGAIGDIQLFKKDKKGREVPISEKDLFDKINGIFSSKPKLEMEDFFIGEFKRYNPSQDLVDQYNIYKKIDSNDNRKKENFYNNHPKFKKDLVRYYYESMCKHEEWEESFEFSKKLQDIGCYVDVNELFTEIETRRLNYKISFCNINADYIDELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQCSLNDITTASANGTQMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQISQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADKDYFEFHIDYAKFTDKAKNSRQIWTICSHGDKRYVYDKTANQNKGAAKGINVNDELKSLFARHHINEKQPNLVMDICQNNDKEFHKSLMYLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNRKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA 13- Leptospira inadai (LiCpfl) (SEQ ID NO: 241)MEDYSGFVNIYSIQKTLRFELKPVGKTLEHIEKKGFLKKDKIRAEDYKAVKKIIDKYHRAYIEEVFDSVLHQKKKKDKTRFSTQFIKEIKEFSELYYKTEKNIPDKERLEALSEKLRKMLVGAFKGEFSEEVAEKYKNLFSKELIRNEIEKFCETDEERKQVSNFKSFTTYFTGFHSNRQNIYSDEKKSTAIGYRIIHQNLPKFLDNLKIIESIQRRFKDFPWSDLKKNLKKIDKNIKLTEYFSIDGFVNVLNQKGIDAYNTILGGKSEESGEKIQGLNEYINLYRQKNNIDRKNLPNVKILFKQILGDRETKSFIPEAFPDDQSVLNSITEFAKYLKLDKKKKSIIAELKKFLSSFNRYELDGIYLANDNSLASISTFLFDDWSFIKKSVSFKYDESVGDPKKKIKSPLKYEKEKEKWLKQKYYTISFLNDAIESYSKSQDEKRVKIRLEAYFAEFKSKDDAKKQFDLLERIEEAYAIVEPLLGAEYPRDRNLKADKKEVGKIKDFLDSIKSLQFFLKPLLSAEIFDEKDLGFYNQLEGYYEEIDSIGHLYNKVRNYLTGKIYSKEKFKLNFENSTLLKGWDENREVANLCVIFREDQKYYLGVMDKENNTILSDIPKVKPNELFYEKMVYKLIPTPHMQLPRIIFSSDNLSIYNPSKSILKIREAKSFKEGKNFKLKDCHKFIDFYKESISKNEDWSRFDFKFSKTSSYENISEFYREVERQGYNLDFKKVSKFYIDSLVEDGKLYLFQIYNKDFSIFSKGKPNLHTIYFRSLFSKENLKDVCLKLNGEAEMFFRKKSINYDEKKKREGHHPELFEKLKYPILKDKRYSEDKFQFHLPISLNFKSKERLNFNLKVNEFLKRNKDINIIGIDRGERNLLYLVMINQKGEILKQTLLDSMQSGKGRPEINYKEKLQEKEIERDKARKSWGTVENIKELKEGYLSIVIHQISKLMVENNAIVVLEDLNIGFKRGRQKVERQVYQKFEKMLIDKLNFLVFKENKPTEPGGVLKAYQLTDEFQSFEKLSKQTGFLFYVPSWNTSKIDPRTGFIDFLHPAYENIEKAKQWINKFDSIRFNSKMDWFEFTADTRKFSENLMLGKNRVWVICTTNVERYFTSKTANSSIQYNSIQITEKLKELFVDIPFSNGQDLKPEILRKNDAVFFKSLLFYIKTTLSLRQNNGKKGEEEKDFILSPVVDSKGRFFNSLEASDDEPKDADANGAYHIALKGLMNLLVLNETKEENLSRPKWKIKNKDWLEFVWERNRKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA14- Lachnospiraceae bacterium ND2006 (LbCpfl) (SEQ ID NO: 242)MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA15- Porphyromonas crevioricanis (PcCpfl) (SEQ ID NO: 243)MDSLKDFTNLYPVSKTLRFELKPVGKTLENIEKAGILKEDEHRAESYRRVKKIIDTYHKVFIDSSLENMAKMGIENEIKAMLQSFCELYKKDHRTEGEDKALDKIRAVLRGLIVGAFTGVCGRRENTVQNEKYESLFKEKLIKEILPDFVLSTEAESLPFSVEEATRSLKEFDSFTSYFAGFYENRKNIYSTKPQSTAIAYRLIHENLPKFIDNILVFQKIKEPIAKELEHIRADFSAGGYIKKDERLEDIFSLNYYIHVLSQAGIEKYNALIGKIVTEGDGEMKGLNEHINLYNQQRGREDRLPLFRPLYKQILSDREQLSYLPESFEKDEELLRALKEFYDHIAEDILGRTQQLMTSISEYDLSRIYVRNDSQLTDISKKMLGDWNAIYMARERAYDHEQAPKRITAKYERDRIKALKGEESISLANLNSCIAFLDNVRDCRVDTYLSTLGQKEGPHGLSNLVENVFASYHEAEQLLSFPYPEENNLIQDKDNVVLIKNLLDNISDLQRFLKPLWGMGDEPDKDERFYGEYNYIRGALDQVIPLYNKVRNYLTRKPYSTRKVKLNFGNSQLLSGWDRNKEKDNSCVILRKGQNFYLAIMNNRHKRSFENKMLPEYKEGEPYFEKMDYKFLPDPNKMLPKVFLSKKGIEIYKPSPKLLEQYGHGTHKKGDTFSMDDLHELIDFFKHSIEAHEDWKQFGFKFSDTATYENVSSFYREVEDQGYKLSFRKVSESYVYSLIDQGKLYLFQIYNKDFSPCSKGTPNLHTLYWRMLFDERNLADVIYKLDGKAEIFFREKSLKNDHPTHPAGKPIKKKSRQKKGEESLFEYDLVKDRRYTMDKFQFHVPITMNFKCSAGSKVNDMVNAHIREAKDMHVIGIDRGERNLLYICVIDSRGTILDQISLNTINDIDYHDLLESRDKDRQQEHRNWQTIEGIKELKQGYLSQAVHRIAELMVAYKAVVALEDLNMGFKRGRQKVESSVYQQFEKQLIDKLNYLVDKKKRPEDIGGLLRAYQFTAPFKSFKEMGKQNGFLFYIPAWNTSNIDPTTGFVNLFHVQYENVDKAKSFFQKFDSISYNPKKDWFEFAFDYKNFTKKAEGSRSMWILCTHGSRIKNFRNSQKNGQWDSEEFALTEAFKSLFVRYEIDYTADLKTAIVDEKQKDFFVDLLKLFKLTVQMRNSWKEKDLDYLISPVAGADGRFFDTREGNKSLPKDADANGAYNIALKGLWALRQIRQTSEGGKLKLAISNKEWLQFVQERSYEKDKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA 16- Prevotella disiens (PdCpfl) (SEQ ID NO: 244)MENYQEFTNLFQLNKTLRFELKPIGKTCELLEEGKIFASGSFLEKDKVRADNVSYVKKEIDKKHKIFIEETLSSFSISNDLLKQYFDCYNELKAFKKDCKSDEEEVKKTALRNKCTSIQRAMREAISQAFLKSPQKKLLAIKNLIENVFKADENVQHFSEFTSYFSGFETNRENFYSDEEKSTSIAYRLVHDNLPIFIKNIYIFEKLKEQFDAKTLSEIFENYKLYVAGSSLDEVFSLEYFNNTLTQKGIDNYNAVIGKIVKEDKQEIQGLNEHINLYNQKHKDRRLPFFISLKKQILSDREALSWLPDMFKNDSEVIKALKGFYIEDGFENNVLTPLATLLSSLDKYNLNGIFIRNNEALSSLSQNVYRNFSIDEAIDANAELQTFNNYELIANALRAKIKKETKQGRKSFEKYEEYIDKKVKAIDSLSIQEINELVENYVSEFNSNSGNMPRKVEDYFSLMRKGDFGSNDLIENIKTKLSAAEKLLGTKYQETAKDIFKKDENSKLIKELLDATKQFQHFIKPLGTGEEADRDLVFYGDFLPLYEKFEELTLLYNKVRNRLTQKPYSKDKIRCFNKPKLMTGWVDSKTEKSDNGTQYGGYLFRKKNEIGEYDYFLGISSKAQLFRKNEAVIGDYERLDYYQPKANTIYGSAYEGENSYKEDKKRLNKVIIAYIEQIKQTNIKKSIIESISKYPNISDDDKVTPSSLLEKIKKVSIDSYNGILSFKSFQSVNKEVIDNLLKTISPLKNKAEFLDLINKDYQIFTEVQAVIDEICKQKTFIYFPISNVELEKEMGDKDKPLCLFQISNKDLSFAKTFSANLRKKRGAENLHTMLFKALMEGNQDNLDLGSGAIFYRAKSLDGNKPTHPANEAIKCRNVANKDKVSLFTYDIYKNRRYMENKFLFHLSIVQNYKAANDSAQLNSSATEYIRKADDLHIIGIDRGERNLLYYSVIDMKGNIVEQDSLNIIRNNDLETDYHDLLDKREKERKANRQNWEAVEGIKDLKKGYLSQAVHQIAQLMLKYNAIIALEDLGQMFVTRGQKIEKAVYQQFEKSLVDKLSYLVDKKRPYNELGGILKAYQLASSITKNNSDKQNGFLFYVPAWNTSKIDPVTGFTDLLRPKAMTIKEAQDFFGAFDNISYNDKGYFEFETNYDKFKIRMKSAQTRWTICTFGNRIKRKKDKNYWNYEEVELTEEFKKLFKDSNIDYENCNLKEEIQNKDNRKFFDDLIKLLQLTLQMRNSDDKGNDYIISPVANAEGQFFDSRNGDKKLPLDADANGAYNIARKGLWNIRQIKQTKNDKKLNLSISSTEWLDFVREKPYLKKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA17- Porphyromonas macacae (PmCpfl) (SEQ ID NO: 245)MKTQHFFEDFTSLYSLSKTIRFELKPIGKTLENIKKNGLIRRDEQRLDDYEKLKKVIDEYHEDFIANILSSFSFSEEILQSYIQNLSESEARAKIEKTMRDTLAKAFSEDERYKSIFKKELVKKDIPVWCPAYKSLCKKFDNFTTSLVPFHENRKNLYTSNEITASIPYRIVHVNLPKFIQNIEALCELQKKMGADLYLEMMENLRNVWPSFVKTPDDLCNLKTYNHLMVQSSISEYNRFVGGYSTEDGTKHQGINEWINIYRQRNKEMRLPGLVFLHKQILAKVDSSSFISDTLENDDQVFCVLRQFRKLFWNTVSSKEDDAASLKDLFCGLSGYDPEAIYVSDAHLATISKNIFDRWNYISDAIRRKTEVLMPRKKESVERYAEKISKQIKKRQSYSLAELDDLLAHYSEESLPAGFSLLSYFTSLGGQKYLVSDGEVILYEEGSNIWDEVLIAFRDLQVILDKDFTEKKLGKDEEAVSVIKKALDSALRLRKFFDLLSGTGAEIRRDSSFYALYTDRMDKLKGLLKMYDKVRNYLTKKPYSIEKFKLHFDNPSLLSGWDKNKELNNLSVIFRQNGYYYLGIMTPKGKNLFKTLPKLGAEEMFYEKMEYKQIAEPMLMLPKVFFPKKTKPAFAPDQSVVDIYNKKTFKTGQKGFNKKDLYRLIDFYKEALTVHEWKLFNFSFSPTEQYRNIGEFFDEVREQAYKVSMVNVPASYIDEAVENGKLYLFQIYNKDFSPYSKGIPNLHTLYWKALFSEQNQSRVYKLCGGGELFYRKASLHMQDTTVHPKGISIHKKNLNKKGETSLFNYDLVKDKRFTEDKFFFHVPISINYKNKKITNVNQMVRDYIAQNDDLQIIGIDRGERNLLYISRIDTRGNLLEQFSLNVIESDKGDLRTDYQKILGDREQERLRRRQEWKSIESIKDLKDGYMSQVVHKICNMVVEHKAIVVLENLNLSFMKGRKKVEKSVYEKFERMLVDKLNYLVVDKKNLSNEPGGLYAAYQLTNPLFSFEELHRYPQSGILFFVDPWNTSLTDPSTGFVNLLGRINYTNVGDARKFFDRFNAIRYDGKGNILFDLDLSRFDVRVETQRKLWTLTTFGSRIAKSKKSGKWMVERIENLSLCFLELFEQFNIGYRVEKDLKKAILSQDRKEFYVRLIYLFNLMMQIRNSDGEEDYILSPALNEKNLQFDSRLIEAKDLPVDADANGAYNVARKGLMVVQRIKRGDHESIHRIGRAQWLRYVQEGIVEKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPD YA

Example 15: Computational Analysis of the Cpf1 Structure

Computational analysis of the primary structure of Cpf1 nucleasesreveals three distinct regions (FIG. 109 ). First a C-terminal RuvC likedomain, which is the only functional characterized domain. Second aN-terminal alpha-helical region and thirst a mixed alpha and betaregion, located between the RuvC like domain and the alpha-helicalregion.

Several small stretches of unstructured regions are predicted within theCpf1 primary structure. Unstructured regions, which are exposed to thesolvent and not conserved within different Cpf1 orthologs, are preferredsides for splits and insertions of small protein sequences. In addition,these sides can be used to generate chimeric proteins between Cpf1orthologs.

Example 16: Generation of Cpf1 Mutants with Enhanced Specificity

Recently a method was described for the generation of Cas9 orthologswith enhanced specificity (Slaymaker et al. 2015). This strategy can beused to enhance the specificity of Cpf1 orthologs.

Primary residues for mutagenesis are all positive charges residueswithin the RuvC domain, since this is the only known structure in theabsence of a crystal and we know that specificity mutants in RuvC workedin Cas9 (see Table below: Conserved Lysine and Arginine residues withinRuvC).

Without wishing to be bound by theory, positively charged residues ofthis region of Cpf1 may act to stabilize the interaction between enzymeand DNA by interacting with the negatively-charged phosphodiesterbackbone of the non-target strand of DNA. By substitution of positivelycharged residues of Cpf1, interactions with the non-target strand may bedisrupted. Sufficient disruption of this interaction can maintainappropriate activity towards target sites but reduce the activity of theenzyme towards non-target sites (which will ordinarily be expected tohave weaker interactions with the guide sequence on account of one ormore mismatches compared the target sequence).

Other domains display similar features. A region of interest is the REC1domain, including but not limited to mutation of one or more amino acidresidues analogous to N497, R661, Q695, and Q926, of SpCas9, andincluding but not limited to mutations to alanine at those positions.Mutations at such residues also disrupt enzyme-DNA phosphate backboneinteractions. Furthermore, combinations of mutations located in the sameor different domains can be employed.

TABLE Conserved Lysine and Arginine residues within RuvC. AsCpf1 LbCpf1R912 R833 T923 R836 R947 K847 K949 K879 R951 K881 R955 R883 K965 R887K968 K897 K1000 K900 R1003 K932 K1009 R935 K1017 K940 K1022 K948 K1029K953 K1072 K960 K1086 K984 F1103 K1003 R1226 K1017 R1252 R1033 R1138R1165Additional candidates are positive charged residues that are conservedbetween different orthologs are provided in the Table below.

TABLE Conserved Lysine and Arginine residues Residue AsCpf1 FnCpf1LbCpf1 MbCpf1 Lys K15 K15 K15 K14 Arg R18 R18 R18 R17 Lys/Arg K26 K26K26 R25 Lys/Arg Q34 R34 K34 K33 Arg R43 R43 R43 M42 Lys K48 K48 K48 Q47Lys K51 K51 K51 K50 Lys/Arg R56 K56 R56 D55 Lys/Arg R84 K87 K83 K85Lys/Arg K85 K88 K84 N86 Lys/Arg K87 D90 R86 K88 Arg N93 K96 K92 K94Lys/Arg R103 K106 R102 R104 Lys N104 K107 K103 K105 Lys T118 K120 K116K118 Lys/Arg K123 Q125 K121 K123 Lys K134 K143 — K131 Arg R176 R186 R158R174 Lys K177 K187 E159 K175 Arg R192 R202 R174 R190 Lys/Arg K200 K210R182 R198 Lys K226 K235 K206 I221 Lys K273 K296 K251 K267 Lys K275 K298K253 Q269 Lys T291 K314 K269 K285 Lys/Arg R301 K320 K271 K291 Lys K307K326 K278 K297 Lys K369 K397 P342 K357 Lys S404 K444 K380 K403 Lys/ArgV409 K449 R385 K409 Lys K414 E454 K390 K414 Lys K436 A483 K415 K448 LysK438 E491 K421 K460 Lys K468 K527 K457 K501 Lys D482 K541 K471 K515 LysK516 K581 A506 K550 Arg R518 R583 R508 R552 Lys K524 K589 K514 K558 LysK530 K595 K520 K564 Lys K532 K597 K522 K566 Lys K548 K613 K538 K582 LysK559 K624 Y548 K593 Lys K570 K635 K560 K604 Lys/Arg R574 K639 K564 K608Lys K592 K656 K580 K623 Lys D596 K660 K584 K627 Lys K603 K667 K591 K633Lys K607 K671 K595 K637 Lys K613 K677 K601 E643 Lys C647 K719 K634 K780Lys/Arg R681 K725 K640 Y787 Lys/Arg K686 K730 R645 K792 Lys H720 K763K679 K830 Lys K739 K782 K689 Q846 Lys K748 K791 K707 K858 Lys/Arg K757R800 T716 K867 Lys/Arg T766 K809 K725 K876 Lys/Arg K780 K823 R737 K890Arg R790 R833 R747 R900 Lys/Arg P791 K834 R748 K901 Lys K796 K839 K753M906 Lys K809 K852 K768 K921 Lys K815 K858 K774 K927 Lys T816 K859 K775K928 Lys K860 K869 K785 K937 Lys/Arg R862 K871 K787 K939 Arg R863 R872R788 R940 Lys K868 K877 Q793 K945 Lys K897 K905 K821 Q975 Arg R909 R918R833 R987 Arg R912 R921 R836 R990 Lys T923 K932 K847 K1001 Lys/Arg R947I960 K879 R1034 Lys K949 K962 K881 I1036 Arg R951 R964 R883 R1038 ArgR955 R968 R887 R1042 Lys K965 K978 K897 K1052 Lys K968 K981 K900 K1055Lys K1000 K1013 K932 K1087 Arg R1003 R1016 R935 R1090 Lys K1009 K1021K940 K1095 Lys K1017 K1029 K948 N1103 Lys K1022 K1034 K953 K1108 LysK1029 K1041 K960 K1115 Lys A1053 K1065 K984 K1139 Lys K1072 K1084 K1003K1158 Lys/Arg K1086 K1098 K1017 R1172 Lys/Arg F1103 K1114 R1033 K1188Lys S1209 K1201 K1121 K1276 Arg R1226 R1218 R1138 R1293 Arg R1252 R1244R1165 A1319 Lys K1273 K1265 K1190 K1340 Lys K1282 K1274 K1199 K1349 LysK1288 K1281 K1208 K1356

The Table above provides the positions of conserved Lysine and Arginineresidues in an alignment of Cpf1 nuclease from Francisella novicida U112(FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1), Lachnospiraceae bacteriumND2006 (LbCpf1) and Moraxella bovoculi 237 (MbCpf1). These can be usedto generate Cpf1 mutants with enhanced specificity.

Example 17: Improving Specificity of Cpf1 Binding

With a similar strategy used to improve Cas9 specificity, specificity ofCpf1 can be improved by mutating residues that stabilize thenon-targeted DNA strand. This may be accomplished without a crystalstructure by using linear structure alignments to predict 1) whichdomain of Cpf1 binds to which strand of DNA and 2) which residues withinthese domains contact DNA.

However, this approach may be limited due to poor conservation of Cpf1with known proteins. Thus it may be desirable to probe the function ofall likely DNA interacting amino acids (lysine, histidine and arginine).

Positively charged residues in the RuvC domain are more conservedthroughout Cpf1s than those in the Rad50 domain indicating that RuvCresidues are less evolutionarily flexible. This suggests that rigidcontrol of nucleic acid binding is needed in this domain (relative tothe Rad50 domain). Therefore, it is possible this domain cuts thetargeted DNA strand because of the requirement for RNA:DNA duplexstabilization (precedent in Cas9). Furthermore, more arginines arepresent in the RuvC domain (5% of RuvC residues 904 to 1307 vs 3.8% inthe proposed Rad50 domains) suggesting again that RuvC targets one ofthe DNA strands. Arginines are more involved in binding nucleic acidmajor and minor grooves (Rohs Nature 2009:http://rohslab.cmb.usc.edu/Papers/Rohs_etal_Nature.pdf). Major/minorgrooves would only be present in a duplex (such as DNA:RNA targetingduplex), further suggesting that RuvC may be involved in cutting.

FIGS. 110, 111 and 112 and provide crystal structures of two similardomains as those found in Cpf1 (RuvC holiday junction resolvase andRad50 DNA repair protein). Based on these structures, it can be deducedwhat the relevant domains look like in Cpf1, and infer which regions andresidues may contact DNA. In each structure residues are highlightedthat contact DNA. In the alignments in FIG. 113 the regions of AsCpf1that correspond to these DNA binding regions are annotated. The list ofresidues in Table below are those found in the two binding domains.

TABLE list of probable DNA interacting residues RuvC domain probableRad50 domain probable DNA interacting residues: DNA interactingresidues: AsCpf1 AsCpf1 R909 K324 R912 K335 R930 K337 R947 R331 K949K369 R951 K370 R955 R386 K965 R392 K968 R393 K1000 K400 K1002 K404 R1003K406 K1009 K408 K1017 K414 K1022 K429 K1029 K436 K1035 K438 K1054 K459K1072 K460 K1086 K464 R1094 R670 K1095 K675 K1109 R681 K1118 K686 K1142K689 K1150 R699 K1158 K705 K1159 R725 R1220 K729 R1226 K739 R1242 K748R1252 K752 R670

From these specific observations about AsCpf1 we can identify similarresidues in Cpf1 from other species by sequence alignments. Examplegiven in FIG. 114 of AsCpf1 and FnCpf1 aligned, identifying Rad50binding domains and the Arginines and Lysines within.

Example 18: Multiplexing with Cpf1 Using Tandem Guides

It was considered whether multiplexing is possible with the Cpf1 enzyme.For this purpose, guide RNAs were developed whereby different guidesequences were positioned in tandem under the same promoter, and theability of these guides to direct genome editing to their respectivetargets was determined.

150,000 HEK293T cells were plated per 24-well 24 h before transfection.Cells were transfected with 400 ng huAsCpf1 plasmid and 100 ng of tandemguide plasmid comprising one guide sequence directed to GRIN28 and onedirected to EMX1 placed in tandem behind the U6 promoter (FIG. 115A),using Lipofectamin2000. Cells were harvested 72 h after transfection andAsCpf1 activity mediated by tandem guides was assayed using the SURVEYORnuclease assay.

The results are demonstrated in FIG. 115B, which demonstrates INDELformation in both the GRIN28 and the EMX1 gene.

It was thus determined that AsCpf1 and by analogy LbCpf1 can employ twoguides expressed from the same U6 promoter without loss in activity. Theposition within the tandem has no influence on the indel formation. Thisdemonstrated that Cpf1 can be used for multiplexing using two or moreguides.

The invention is further described by the following numbered paragraphs:

-   -   1. An engineered, non-naturally occurring Clustered Regularly        Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated        (Cas) (CRISPR-Cas) system comprising        -   a) one or more Type V CRISPR-Cas polynucleotide sequences            comprising a guide RNA which comprises a guide sequence            linked to a direct repeat sequence, wherein the guide            sequence is capable of hybridizing with a target sequence,            or one or more nucleotide sequences encoding the one or more            Type V CRISPR-Cas polynucleotide sequences, and        -   b) a Cpf1 effector protein, or one or more nucleotide            sequences encoding the Cpf1 effector protein;        -   wherein the one or more guide sequences hybridize to said            target sequence, said target sequence is 3′ of a Protospacer            Adjacent Motif (PAM), and said guide RNA forms a complex            with the Cpf1 effector protein.    -   2. An engineered, non-naturally occurring Clustered Regularly        Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated        (Cas) (CRISPR-Cas) vector system comprising one or more vectors        comprising        -   c) a first regulatory element operably linked to one or more            nucleotide sequences encoding one or more Type V CRISPR-Cas            polynucleotide sequences comprising a guide RNA which            comprises a guide sequence linked to a direct repeat            sequence, wherein the guide sequence is capable of            hybridizing with a target sequence,        -   d) a second regulatory element operably linked to a            nucleotide sequence encoding a Cpf1 effector protein;        -   wherein components (a) and (b) are located on the same or            different vectors of the system,        -   wherein when transcribed, the one or more guide sequences            hybridize to said target sequence, said target sequence is            3′ of a Protospacer Adjacent Motif (PAM), and said guide RNA            forms a complex with the Cpf1 effector protein.    -   3. The system of numbered paragraph 1 or 2 wherein the target        sequences is within a cell.    -   4. The system of numbered paragraph 3 wherein the cell comprises        a eukaryotic cell.    -   5. The system according to any one of paragraphs 1-4, wherein        when transcribed the one or more guide sequences hybridize to        the target sequence and the guide RNA forms a complex with the        Cpf1 effector protein which causes cleavage distally of the        target sequence.    -   6. The system according to numbered paragraph 5, wherein said        cleavage generates a staggered double stranded break with a 4 or        5-nt 5′ overhang.    -   7. The system according to any one of numbered paragraphs 1-6,        wherein the PAM comprises a 5′ T-rich motif.    -   8. The system according to any one of numbered paragraphs 1-7,        wherein the effector protein is a Cpf1 effector protein derived        from a bacterial species listed in FIG. 64 .    -   9. The system according to numbered paragraph 8, wherein the        Cpf1 effector protein is derived from a bacterial species        selected from the group consisting of Francisella tularensis 1,        Francisella tularensis subsp. novicida, Prevotella albensis,        Lachnospiraceae bacterium MC2017 1, Butyrivibrio        proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10,        Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,        Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020,        Candidatus Methanoplasma termitum, Eubacterium eligens,        Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae        bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella        disiens and Porphyromonas macacae.    -   10. The system according to numbered paragraph 9, wherein the        PAM sequence is TTN, where N is A/C/G or T and the effector        protein is FnCpf1 or wherein the PAM sequence is TTTV, where V        is A/C or G and the effector protein is PaCpf1p, LbCpf1 or        AsCpf1.    -   11. The system of any one of numbered paragraphs 1-10, wherein        the Cpf1 effector protein comprises one or more nuclear        localization signals.    -   12. The system of any one of numbered paragraphs 1-11, wherein        the nucleic acid sequences encoding the Cpf1 effector protein is        codon optimized for expression in a eukaryotic cell.    -   13. The system of any one of numbered paragraphs 1-12 wherein        components (a) and (b) or the nucleotide sequences are on one        vector.    -   14. A method of modifying a target locus of interest comprising        delivering a system of any one of numbered paragraphs 1-13, to        said locus or a cell containing the locus.    -   15. A method of modifying a target locus of interest, the method        comprising delivering to said locus a non-naturally occurring or        engineered composition comprising a Cpf1 effector protein and        one or more nucleic acid components, wherein the Cpf1 effector        protein forms a complex with the one or more nucleic acid        components and upon binding of the said complex to a target        locus of interest that is 3′ of a Protospacer Adjacent Motif        (PAM), the effector protein induces a modification of the target        locus of interest, wherein the complex comprises Mg²⁺.    -   16. The method of numbered paragraph 14 or 15, wherein the        target locus of interest is within a cell.    -   17. The method of numbered paragraph 16, wherein the cell is a        eukaryotic cell.    -   18. The method of numbered paragraph 16, wherein the cell is an        animal or human cell.    -   19. The method of numbered paragraph 16, wherein the cell is a        plant cell.    -   20. The method of numbered paragraph 14 or 15, wherein the        target locus of interest is comprised in a DNA molecule in        vitro.    -   21. The method of any one of numbered paragraphs 15-20, wherein        said non-naturally occurring or engineered composition        comprising a Cpf1 effector protein and one or more nucleic acid        components is delivered to the cell as one or more        polynucleotide molecules.    -   22. The method of any one of numbered paragraphs 14-21, wherein        the target locus of interest comprises DNA.    -   23. The method of numbered paragraph 22, wherein the DNA is        relaxed or supercoiled.    -   24. The method of any one of numbered paragraphs 14-23, wherein        the composition comprises a single nucleic acid component.    -   25. The method of numbered paragraph 24, wherein the single        nucleic acid component comprises a guide sequence linked to a        direct repeat sequence.    -   26. The method of any one of numbered paragraphs 14-25 wherein        the modification of the target locus of interest is a strand        break.    -   27. The method of numbered paragraph 26, wherein the strand        break comprises a staggered DNA double stranded break with a 4        or 5-nt 5′ overhang.    -   28. The method of numbered paragraph 26 or 27, wherein the        target locus of interest is modified by the integration of a DNA        insert into the staggered DNA double stranded break.    -   29. The method of any one of numbered paragraphs 14-28, wherein        the Cpf1 effector protein comprises one or more nuclear        localization signal(s) (NLS(s)).    -   30. The method of any one of numbered paragraphs 21-29, wherein        the one or more polynucleotide molecules are comprised within        one or more vectors.    -   31. The method of any one of numbered paragraphs 21-30, wherein        the one or more polynucleotide molecules comprise one or more        regulatory elements operably configured to express the Cpf1        effector protein and/or the nucleic acid component(s),        optionally wherein the one or more regulatory elements comprise        inducible promoters.    -   32. The method of any one of numbered paragraphs 21 to 31        wherein the one or more polynucleotide molecules or the one or        more vectors are comprised in a delivery system.    -   33. The method of any one of numbered paragraphs 14-30, wherein        system or the one or more polynucleotide molecules are delivered        via particles, vesicles, or one or more viral vectors.    -   34. The method of numbered paragraph 33 wherein the particles        comprise a lipid, a sugar, a metal or a protein.    -   35. The method of numbered paragraph 33 wherein the vesicles        comprise exosomes or liposomes.    -   36. The method of numbered paragraph 33 wherein the one or more        viral vectors comprise one or more of adenovirus, one or more        lentivirus or one or more adeno-associated virus.    -   37. The method of any one of numbered paragraphs 14-36, which is        a method of modifying a cell, a cell line or an organism by        manipulation of one or more target sequences at genomic loci of        interest.    -   38. A cell from the method of numbered paragraph 37, or progeny        thereof, wherein the cell comprises a modification not present        in a cell not subjected to the method.    -   39. The cell of numbered paragraph 38, of progeny thereof,        wherein the cell not subjected to the method comprises an        abnormality and the cell from the method has the abnormality        addressed or corrected.    -   40. A cell product from the cell or progeny thereof of numbered        paragraph 38, wherein the product is modified in nature or        quantity with respect to a cell product from a cell not        subjected to the method.    -   41. The cell product of numbered paragraph 40, wherein the cell        not subjected to the method comprises an abnormality and the        cell product reflects the abnormality having been addressed or        corrected by the method.    -   42. An in vitro, ex vivo or in vivo host cell or cell line or        progeny thereof comprising a system of any one of numbered        paragraphs 1-13.    -   43. The host cell or cell line or progeny thereof according to        numbered paragraph 42, wherein the cell is a eukaryotic cell.    -   44. The host cell or cell line or progeny thereof according to        numbered paragraph 43, wherein the cell is an animal cell.    -   45. The host cell or cell line or progeny thereof of numbered        paragraph 33, wherein the cell is a human cell.    -   46. The host cell, cell line or progeny thereof according to        numbered paragraph 31 comprising a stem cell or stem cell line.    -   47. The host cell or cell line or progeny thereof according to        numbered paragraph 30, wherein the cell is a plant cell.    -   48. A method of producing a plant, having a modified trait of        interest encoded by a gene of interest, said method comprising        contacting a plant cell with a system according to any one of        numbered paragraphs 1-13 or subjecting the plant cell to a        method according to numbered paragraph 14-17 or 19 to 37,        thereby either modifying or introducing said gene of interest,        and regenerating a plant from said plant cell.    -   49. A method of identifying a trait of interest in a plant, said        trait of interest encoded by a gene of interest, said method        comprising contacting a plant cell with a system according to        any one of numbered paragraphs 1-13 or subjecting the plant cell        to a method according to numbered paragraph 14-17 or 19 to 37,        thereby identifying said gene of interest.    -   50. The method of numbered paragraphs 49, further comprising        introducing the identified gene of interest into a plant cell or        plant cell line or plant germplasm and generating a plant        therefrom, whereby the plant contains the gene of interest.    -   51. The method of numbered paragraph 50 wherein the plant        exhibits the trait of interest.    -   52. A particle comprising a system according to any one of        numbered paragraphs 1-13.    -   53. The particle of numbered paragraph 52, wherein the particle        contains the Cpf1 effector protein complexed with the guide RNA.    -   54. The system or method of any preceding numbered paragraph,        wherein the complex, guide RNA or protein is conjugated to at        least one sugar moiety, optionally N-acetyl galactosamine        (GalNAc), in particular triantennary GalNAc.    -   55. The system or method of any preceding numbered paragraph,        wherein the concentration of Mg²⁺ is about 1 mM to about 15 mM.    -   56. An isolated protein having at least 60% sequence identity        with AsCpf1 or LbCpf1, and capable of binding a target DNA        through a complex with a guide RNA comprising a direct repeat        sequence and a guide sequence, without requiring the presence of        a tracrRNA.    -   57. An isolated nucleic acid encoding a protein according to        numbered paragraph 56.    -   58. The method of numbered paragraph 17, which is a method of        treatment of a disease caused by a genetic defect in said cell.    -   59. The method of numbered paragraph 58, wherein said method is        carried out on a cell in vivo or ex vivo.    -   60. A non-naturally occurring or engineered composition        comprising a Cpf1 effector protein and one or more guide RNA        comprising a direct repeat sequence and a guide sequence capable        of hybridizing to a target DNA at a locus of interest, wherein        the Cpf1 effector protein forms a complex with the one or more        guide RNAs and upon binding of the said complex to a target        locus of interest that is 3′ of a Protospacer Adjacent Motif        (PAM), the effector protein induces a modification of the target        locus of interest.    -   61. A non-naturally occurring or engineered composition        comprising a polynucleotide sequence encoding a Cpf1 effector        protein and one or more guide RNA comprising a direct repeat        sequence and a guide sequence capable of hybridizing to a target        DNA at a locus of interest, wherein the Cpf1 effector protein,        when expressed, forms a complex with the one or more guide RNAs        and upon binding of the said complex to a target locus of        interest that is 3′ of a Protospacer Adjacent Motif (PAM), the        effector protein induces a modification of the target locus of        interest.    -   62. The composition according to numbered paragraph 60 or 61        which is a pharmaceutical composition.    -   63. The composition according to numbered paragraph 60 or 61,        for use as a medicament.    -   64. The composition according to numbered paragraph 60 or 61 for        use in the treatment of a disease or disorder caused by a        genetic defect at the target locus of interest.    -   65. The method according to numbered paragraph 58, or the        composition for use according to statement 64, wherein the cell        is a HSC cell.    -   66. The method according to numbered paragraph 58, or the        composition for use according to statement 64, wherein the        disease or disorder is a blood cell disorder.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed:
 1. A CRISPR-Cas system comprising, a Type V Caspolypeptide and one or more engineered nucleic acid components that forma CRISPR-Cas complex with the Type V Cas polypeptide and that is capableof directing sequence-specific binding of said complex to a targetsequence present in a eukaryotic cell.
 2. The CRISPR-Cas system of claim1, wherein the Type V Cas polypeptide comprises a RuvC-like nucleasedomain, but does not comprise an HNH domain.
 3. The CRISPR-Cas system ofclaim 1, wherein the Type V Cas polypeptide is a Type V-A Caspolypeptide.
 4. The CRISPR-Cas system of claim 1, wherein the Type V Caspolypeptide comprises at least one mutation in the RuvC-like nucleasedomain and is catalytically inactive.
 5. The CRISPR-Cas system of claim1, wherein the Type V Cas polypeptide is catalytically active.
 6. TheCRISPR-Cas system of claim 1, wherein the Type V Cas polypeptide isconfigured to cleave only one strand at the location of the targetsequence.
 7. The CRISPR-Cas system of claim 1, wherein the Type V Caspolypeptide comprises a heterologous functional domain.
 8. A compositioncomprising a complex, which complex comprises a Type V Cas polypeptideand one or more engineered nucleic acid components, wherein the Type VCas polypeptide and the one more engineered nucleic acid components donot naturally occur together.
 9. The composition of claim 8, wherein thecomplex comprises a Type V Cas polypeptide comprising one or morecatalytic motifs of a RuvC nuclease domain but not comprising a HNHdomain.
 10. The composition of claim 8, wherein the complex comprises(a) a Type V Cas polypeptide comprising one or more catalytic motifs ofthe RuvC nuclease domain and a Zn finger region, but not comprising aHNH domain, and (b) one or more nucleic acid components.
 11. Thecomposition of claim 10, wherein the Zn finger region is inactivated.12. The composition of claim 8, wherein the Type V Cas polypeptide is aType V-A Cas polypeptide.
 13. The composition of claim 8, wherein theType V Cas polypeptide comprises at least one mutation in the RuvC-likenuclease domain and is catalytically inactive.
 14. The composition ofclaim 8, wherein the Type V Cas polypeptide is catalytically active. 15.The composition of claim 8, wherein the Type V Cas polypeptide isconfigured to cleave only one strand at the location of a targetsequence.
 16. The composition of claim 8, wherein the Type V Caspolypeptide comprises a heterologous functional domain.
 17. A eukaryoticcell comprising: a Cas polypeptide that comprises a RuvC-like nucleasedomain, but does not comprise an HNH domain; and one or more engineerednucleic acid components that form a CRISPR-Cas complex with the Caspolypeptide and that is capable of directing sequence-specific bindingof said complex to a target sequence present in the eukaryotic cell. 18.The eukaryotic cell of claim 17, wherein the Cas polypeptide is a Type VCas polypeptide.
 19. The eukaryotic cell of claim 17, in which thetarget sequence is cleaved.
 20. The eukaryotic cell of claim 17, whereinthe one or more nucleic acid components are engineered to hybridize witha target sequence adjacent to a protospacer motif (PAM) in the genome ofthe eukaryotic cell.
 21. The eukaryotic cell of claim 17, wherein theCas polypeptide comprises one or more nuclear localization signalsand/or the one or more nucleic acid components comprise one or moremodified nucleotides or one or more non-nucleotide components.
 22. Theeukaryotic cell of claim 17, which comprises the formed CRISPR-Cascomplex.
 23. A method of targeting a polynucleotide, comprisingcontacting a sample comprising the polynucleotide with a CRISPR-Cascomplex comprising, 1) a Cas polypeptide comprising a RuvC nucleasedomain, but not comprising a HNH domain, and 2) one or more nucleotidecomponents capable of directing sequence-specific binding of theCRISPR-Cas complex to a target sequence of the polynucleotide.
 24. Themethod of claim 23, wherein the Cas polypeptide is a Type V Caspolypeptide.
 25. The method of claim 23, wherein the Cas polypeptidecomprises at least one mutation in the RuvC-like nuclease domain and iscatalytically inactive.
 26. The method of claim 23, wherein the Caspolypeptide is catalytically active.
 27. The method of claim 23, whereinthe Cas polypeptide is configured to cleave only one strand at thelocation of the target sequence.
 28. The method of claim 23, wherein thetarget sequence is in a eukaryotic cell.
 29. The method of claim 28,which comprises contacting the eukaryotic cell with the CRISPR-Cascomplex and results in modification of a gene or gene product, ormodification in the expression of a gene product.
 30. The method ofclaim 28, which comprises contacting the eukaryotic cell with theCRISPR-Cas complex and results in cleavage of the target sequence.